0020-711X/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc
Inf.J. Biochem.Vol. 23, No. 4, pp. 421-428, 1991 Printed in Great Britain. All rights reserved
PURIFICATION AND CHARACTERIZATION OF HUMAN TESTIS ALDOSE AND ALDEHYDE REDUCTASE TSUYOSHITANIMOTO,’MIYAKOOHTA,’AKIRATANAKA,’ISAOIKEMOTO’and TOYOHEIMACHIDA* ‘Division of Biological Chemistry, National Institute of Hygienic Sciences, l-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan [Tel. 700-I 1411 *Department of Urology, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan (Rem-bed 6 June l?WU)
Abstract-I.
Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pl values of these enzymes were found to be 5.9 and 5.1 by chromatof~using, respectively. 3. Aidose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The K,,, values of aldose reductase for o-glucose, D-galactose and D-XylOSe were 57, 49 and 6.2mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 1O-7-1O-g M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5’-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.
INTRODWCTION Aldose reductase (alditol : NADP+ I-oxidoreductase, EC 1.1.1.2 1) catalyzes the first reaction of the polyol pathway, and the enzyme catalyzes the conversion of aldose to the corresponding sugar-alcohol. Aldose reductase has been found to be present in various mammalian tissues (Gabbay, 1975; Kern and Engerman, 1982; Markus et al., 1983; Srivastava et al., 1984). However, its metabolic significance has only been studied in the seminal vesicles. Ludvigson and Sorenson (1980) demonstrated the presence of aldose reductase in rat testes using immunohist~hemi~l techniques. It has been suggested that testis aldose reductase together with sorbitol dehydrogenase constitutes the polyol pathway and may be responsible for the formation of D-fructose which has a trophic role for spermatides in late stages of s~~atogenesis. In recent years, interest in this enzyme has increased because of its possible role in the pathogenesis of diabetic complications such as cataract, retinopa~y, neuropathy and nephropathy (Buszney et al., 1977; Kador et al., 1979; Kador et al., 1985; Kinoshita, 1988). Indeed, some aldose reductase inhibitors have been found to be useful for preventing or treating chronic complications due to diabetes (Jed~wi~h et al., 1983; Jaspan et al., 1983). However, there are no studies with regard to the effect of these inhibitors on aldose reductase in tissues which are not directly associated with diabetic complications. A knowledge of the enzymatic properties and susceptibility to inhibition of aldose reductase in
tissues unaccompanied by diabetic complications would be useful for the elucidation of the side effects of aldose reductase inhibitors. In this paper, we report for the first time the purification, characterization and susceptibility to inhibition by commercially developed aldose reductase inhibitors of human testis aldose reductase and aldehyde reductase. MATERIALSAND METHODS Materials DL-Gly~raldehyde and rr-glucuronic acid were purchased from Aidrich Chemical Co: and the other aldbses from Wako Pure Chemical Industries Ltd. NADPH and NADH were obtained from Oriental Yeast Co. Sephadex G-75, Polybuffer 74 and Polybuffer exchanger 94 were purchased from Pharmacia Fine Chemicals. Molecular weight standard protein was obtained from Bio-Rad Laboratories. Matrex gel orange A was purchased from Amicon Co. Sorbinil [(S)-6-fluorospiro-(chroman-4,5’-imidazolidme)2’,4’-dione], tolrestat (N-[(S-tri-fluoro-methyl-6-methoxyI-naphthaIenyi)thioxomethyl]-~-methyl-~ycine), M79175 [2-methyl-6-fluorospiro-(chroman-4,5’-imidazolidine-~,~dione)], ponalrestat ([3-(4-bromo-2-fluorobenzyl)-4-oxo3H-phthalazin-l-yl]-acetic acid) and AL1576 (2,7-difluorospirofluorene-9,5’-imidazolidine-2’,4’-dione) were kindly donated by Dr Kador, National Institutes of Health. U.S.A. Epalrestat ((E,E)-5-[(2-methyl-3-phenyi-2-pro~nyiidene)4-oxo-2-thioxo-3-thiazolidinel-acetic acid) was a gift of Professor Tanaka, Osaka University of Pharmaceutical Sciences. The protein assay kit was obtained from Bio-Rad Laboratories. Testes were obtained from human and stored frozen at -80°C.
421
422
Tsuvowr
TANIMOKJ et al.
Table 1. Purification of aldose reductase and aldehvde reductase from human testis
Step Extraction Matrex gel orange A Sephadex G-75 Chromatofocusing Aldose reductase (peak I) Aldehyde reductase (peak II)
Total protein (mg)
Total activity (U)
Sp. act. (Wmg)
2886 8.32 5.85
4.65 3.51 3.41
0.0016 0.429 OS83
0.916 1.030
1.32 2.66
0.694 0.387
Assay of enzyme activity
The activities of aldose reductase and aldehyde reductase were determined at 25°C by measuring the decrease in absorption of NADPH at 340nm on a Hitachi U-2090 spectrophotometer equipped with a temperature-controlled cuvette chamber. Assay mixture in a l.Oml system contained 90 mM sodium phosphate buffer (pH 6.2), 0.15 mM NADPH and 10mM m-glyceraldehyde. The reaction was initiated by adding the enzyme, and the rate of NADPH oxidation was followed by recording the decrease in absorbance at 340 nm. The appropriate blank for correction of nonspecific oxidation of NADPH was prepared. One unit of the enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 pmol of NADPH per min under the conditions described here. Determination
of kinetic constant
initial rate determination of each substrate was carried out in duplicate at six concentrations, and the values were calculated with a computer using the programs of Cleland (1963). The
Determination
of IC,
The concentration of inhibitor giving 50% inhibition of the enzyme activity (IC,) was estimated from the least squares regression line of the log dose-response plot. Protein
&termination
concentration of protein was determined with the Bio-Rad protein assay kit according to the method of Bradford (1976). The
Polyacrylamide
gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on slab gels containing a 12% acrylamide according to the method of Laemmli (1970). Protein were visualized with Coomassie Brilliant Blue R250. Standard proteins used for estimation of molecular weight were trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa). Enzyme purification
All operations were performed at 0-4”C. All buffers contained 2mM dithiothreitol in order to protect the enzyme from inactivation. Step 1. Extraction: human testis of 12 g was homogenized in 30 ml of 20 mM sodium phosphate buffer @H 7.0) with Polytron, and the homogenate was centrifuged at 15,OOOg for 30 min to remove insoluble materials. Step 2. Matrex gel orange A afinity chromatography: the extract of human testis was diluted to 3-times with 20 mM phosphate buffer (pH 7.0), and was applied directly to a column (1.5 x 25 cm) of Matrex gel orange A equilibrated with 20 mM sodium phosphate buffer @H 7.0). After the column was thoroughly washed with the same buffer, the enzyme was eluted with a linear concentration gradient of sodium chloride, formed from lSOm1 of 20mM sodium phosphate buffer @H 7.0) in the mixing chamber and 150 ml of the same buffer containing 0.6 M sodium chloride in the
Purification (fold)
I
Yield W)
267 364
100 75.5 73.3
825 I663
19.7 22.2
reservoir, at a flow rate of 25 ml per hr. The enzyme activity was observed in the fraction eluted with ca 0.25 M sodium chloride. Fractions with enzyme activities were pooled and concentrated by ultrafiltration using a collodion bag. Step 3. Sephadex G- 7.5 gel jiltration: the concentrated enzyme solution from Step 2 was placed on a columm (1.2 x 50 cm) of Sephadex G-75 equilibrated with 25 mM imidazole_HCl buffer (pH 7.2) and was eluted with the same buffer at a flow rate of 10ml per hr. The enzyme fractions obtained were pooled and concentrated by ultrafiltration using a collodion bag. Step 4. Chromatofocusing: the concentrated enzymes solution described above was sub&ted to chromatofocusinn on a column (1 x 40 cm) of polybuffer exchanger 94 equilc brated with 25 mM imidazoleHC1 buffer (OH 7.2). The elution was carried out with 1:8 diluted polybufi’er 74 (PH 4.5) at a flow rate of 20 ml per hr, and fractions of 2.2 ml were collected. RESULTS AND DISCUSSION Enzyme purification Two NADPH-dependent reductases have been purified from human testis in the four steps summar-
ized in Table 1. With DL-glyceraldehyde as substrate only a single peak of NADPH-dependent reductase activity was obtained from affinity chromatography on Matrex gel orange A and gel filtration on Sephadex G-75. Subsequent chromatofocusing of enzyme fractions obtained from gel filtration resulted in the appearance of two peaks of enzyme activity, tentatively designated as peaks I and II (Fig. 1). On the basis of enzymatic properties of these enzymes described in this paper, the enzymes in peaks I and II were identified as aldose reductase and aldehyde reductase, respectively. Aldose reductase in human testis was purified ca 820-fold with the sp. act. of 1.32 U per mg, and was recovered in ca 20%. Aldehyde reductase was purified ca 1660-fold with a sp. act. of 2.66 U per mg, and the yield was ca 22%. Molecular weights and isoelectric points The molecular weights of human testis aldose reductase and aldehyde reductase were estimated to IX 36,000 and 38,000 by SDS-PAGE respectively
(Fig. 2). The molecular weights of human testis aldose reductase and aldehyde reductase were similar to those reported for both enzymes from various other sources (Clements and Winegrad, 1972; Sheaff and Doughty, 1976; Boghosian and McGuinness, 1979; Herrmann et al., 1983). The p1 values determined by chromatofocusing were 5.9 for aldose reductase and 5.1 for aldehyde reductase. In mammalian tissues such as ox brain (Moonsammy and Stewart, 1967), rat testis (Kawasaki et al., 1989) and EHS tumor cell (Tanimoto et al., 1990), aldose reductase was more acidic protein than aldehyde
423
Human testis aldose and atdehyde reductase
Fraction number
Fig.
1. ~hr~matof~usj~g
of aldose reductase and aldehyde reductase from human testis. The pooled
fractions obtained from gel filtration was applied to a c~romatof~~ing column. Fractions of i Ximl were measured for protein content (---), enzyme activity (a) and pH c.-). reductase. However, the p1 value of aldose reductase in human tissues was higher than that of aldehyde reductase (O’Brien and Schofield, 1980, Srivastava et al., 1984; Das et al., 1985).
The kinetic constants of aldose reductase and aldehyde reductase were determined for several substrates and are given in Table 2. Aldose reductase reduced aldo-sugars such as o-xylose, ~-glucose and D-g$aCtOSe, and the Km values for these substrates were 6.2, 57, 49mM, respectively. Compared to Dt-glyceraldehyde, aldose reductase displayed relatively high K,,, values for D-xylose, D-glucose and D-galactose and significantly low k,,/K, values for these aldo-sugars. However, because the aldehyde form of aldo-sugars is the real substrate for aldose reductase, as pointed out by Inagaki et al. (1932), accounting for the aldehyde form of a-glucose and n-galactose leads to very low corrected K, values (6.9 BM for D-glucose and 34 FM for D-galactose) and high k,,jii, values (89,~O for o-glucose and 20,500 for D-galactose). The apparent X, values for various aldo-sugars of human testis aldose reductase were lower than those of human psoas muscle enzyme (Morjana and Flynn, 1989), and were in the same range as those reported for the enzyme isolated from rabbit muscle (Cromlish and Flynn, 1983a), human erythrocyte (Das et al., 1985), human placenta (Clements and Winegard, 1972) and rat lens Table 2. Substrate
(Herrmann et al., 1983). Aldose reductase can also reduce D-glucuronate as substrate, but this enzyme displayed a Km value 200-times greater and a k,, JX;, value lo-tirn~ less than those for DL-gly~eraidehyde. The &, value of aldehyde reductase for DL-glyceraldehyde was found to be higher than that of aldose reductase. Aldehyde reductase was virtually inactive for aldo-sugars. The Km vafue of aldehyde reductase for D-gfucuronate was similar to the value for Dt-glyceraldehyde, and the k-*/k;, value for D-glucuronate was 1.5 times higher than DL-gly~raldehyde‘ With respect to coenzyme specificity, aldose reductase utilized both NADPH and NADH as coenzymes. However, the enzyme showed a preference for NADPH. This is indicated by the low K,,, value for NADPH (3.2 PM) as compared to that for NADH (250 ~JM), On the other hand, aldehyde reductase exhibited no enzyme activity with NADH as a eofaetor, and utilized only NADPH as a coenzyme, the K,,, being 3.8 /.iM.
The effects of salts on aldose reductase and aldehyde reductase activities are shown in Table 3. The activity of aldose reductase was increased 3-4times in the presence of 0.3 M ammonium sulfate. Similar activation was also observed in the presence of 0.3 M sodium sulfate. However, 0.3 M ammonium chloride and sodium chloride slightly inactivated aldose reductase. Sodium chloride or ammonium chloride
specificities and kinetic constants of human testis aldose reductase and aldehyde reductase
Aldose reductase
AIdehyde reductase
k
xx.-Giyceraldehyde D-xyiOS.5 D-Giuccse
D-Galactose a-Glucuronate NADPH NADH
k
(=?f
Substrate t3.026 f. 0.001 6.2 + 0.43 57.3 + 3.3 49.3 * 1.7 5.05 rt 0.21 0.0032 rf 0.0003 0.25 * 0.01
0.672 0.633 0.614 0.702 0.697
* F f + +
&&n 0.026 0.043 0.035 0.024 0.029
25,850 102 11 14 138 -
3.17 kO.39 >3ow >45oO >4500 4.69 f 0.27 0.0038 & 0.0004 ND
(se‘?!)
&t/K”
1.33_tO.16 2.34t_O.13 -
420 499 -
For the determination of K, values for different substrates, the concentration of NADPH was kept at 0.15 mM. For the determination of K, values for NAD(P)H, DL-gtyceraldehyde concentration was kept at 3 mM for aidose reductase and 30 mM for aldehyde reductase. ND, not detectable.
94K
-
ZOK
-
Fig. 2. SDS-PAGE of the purified aldosc reductase and aldehyde rcductase from human testis. The gel was stained with Coomassie Brilliant Blue R250. Lane S, standard proteins; lane 1, aldose reductase; lane 2, aldehyde reductase.
424
425
Human testis aldose and aldehyde reductase Table 3. Effect of salts on human testis aldose reductase and aldehyde reductase Relative activity (%)
Salt
Aldose reductase
Aldehyde reductase
None
100
100
(NW=4
338 296 84 98
91 98 76 74
Na,SO, NaCl NH,CI
Effects of different salts on the activities of aldose reductase and aldehyde reductase were studied at 0.3 M concentration.
could not be substituted for sodium sulfate or ammonium sulfate, indicating that sulfate ion was the activating species for aldose reductase. The response of aldose reductase to sulfate ion was similar to that of rat (Herrmann et al., 1983), calf (Haymann and Kinoshita, 1965) and rabbit (Tanimoto et al., 1984) lens, sheep seminal vesicle (Hers, 1960), human erythrocyte (Das et af., 1985) and placenta (Clements and Winegrad, 1972) aldose reductase. In contrast to aldose reductase, aldehyde reductase activity was not affected by 0.3 M ammonium sulfate and sodium sulfate. The enzyme, however, was inactivated slightly by 0.3 M ammonium chloride and sodium chloride as well as aldose reductase. Effect of various inhibitors Valproate, which selectively inhibits aldehyde reductase (Whittle and Terner, 1981; Cromlish and Flynn, 1983b) was a much better inhibitor of human testis aldehyde reductase than of aldose reductase. Sodium valproate at 0.5 mM inhibited aldehyde reductase by ca 90%, but aldose reductase was inhibited only very slightly by the same concentration (Fig. 3). The susceptibility of human testis aldose reductase and aldehyde reductase to inhibition by the commercially developed aldose reductase inhibitors such as sorbinil, tolrestat, M79175, ponalrestat, AL1576 and epalrestat, is summarized in Table 4. Human testis aldose reductase was greatly inhibited by these inhibitors at concentrations of the order of 10-7-10-9 M. Inhibition of human testis aldose reductase appeared to be similar to that observed with aldose reductase from rat testis (Kawasaki et al., 1989), and human
testis aldose reductase was inhibited to a greater extent than the enzyme from human erythrocyte (Nakayama et al., 1989). On the other hand, all six compounds were found to inhibit human testis aldehyde reductase appears to be less susceptible to these inhibitors than aldose reductase. The susceptibility to inhibition of human testis aldehyde reductase was almost the same as that of human erythrocyte (Nakayama et al., 1989), rat testis (Kawasaki et al., 1989) and rat kidney (Sato et al., 1988) enzymes. Aldose reductase together with sorbitol dehydrogenase forms a polyol pathway, and by this pathway D-fructose which is a life energy source to spermatozoa is synthesized. It was reported that aldose reductase was localized to the Sertoli cell and mature spermatids in rat testis (Ludvigson and Sorenson, 1980). Therefore, aldose reductase in the testis may play an important trophic role in spermatids in the late stages of spermatogenesis. Although the permeability of drugs from blood to germ ceil was regulated by the blood-testis barrier (Okumura et al., 1975) and it is not yet clear whether administered aldose reductase inhibitor distribute in a germ cell, it can be presumed from the results described in this paper that the administration of aldose reductase inhibitors, which have been developed for the prevention or treatment of chronic complications due to diabetes, may affect the system of human male reproduction. There may be no gain in saying that aldose reductase inhibitors produce undesirable side-effects in clinical use. Effect of pyridoxal S-phosphate on aldose reductase The reaction of aldose reductase from human testis with 250 and 500 PM of pyridoxal 5’-phosphate (PLP) resulted ca 2 to 2.5-fold activation of the enzyme, however, PLP did not affect on aldehyde reductase (Fig. 4). The activation by PLP was also observed for human psoas muscle aldose reductase (Morjana et al., 1989), but not observed for rat testis (Kawasaki et al., 1989) and rabbit lens (Tanimoto et al., 1986) aldose reductase. Therefore, it may be specific for human aldose reductase that the enzyme is activated by PLP. Although native aldose reductase exhibited monophasic kinetics with DL-glyceraldehyde (K,,, = 0.026 mM), the PLP-activated enzyme
500,
60 Time (min)
Sodium valproate
(mM)
Fig. 3. Inhibition of human testis aldose reductase (0) and aldehyde reductase (m) by sodium valporate. The enzyme activities were assayed in the presence of the indicated concentration of sodium valporate.
Fig. 4. Effect of PLP on human testis aldose reductase and aldehyde reductase. The enzyme (1 x 10e6M) was incubated at 25°C with PLP at the following concentrations: 500 p M (0) and 250 p M (A) for aldose reductase and
500 PM (W) for aldehyde reductase. Aliquots were removed at the various times and were assayed for enzyme activities.
426
TSUKISHITANIMOTO et
of.
Table 4. Effeot of aldose reductase inhibitors on human testis aldose reductase and aldehyde redwtase IC,
tiM)
Aldose reductase
Aldehyde reductase
Inhibitor
HT
HF
RF
RL’
Sotbinil
0.55
1.8
0.18
0.07
M79175
0.044
1.4
0.053
0.055
0.017
AL1576
HT
HE”
RTb
RK’
4.6
7.6
1.5
1.8
0.028
0.16
0.71
0.10
0.16
0.024
0.01 I
0.025
0.012
0.046
1.2
0.50
0.24
0.013
0.010
0.011
0.75
Ponahestat
0.13
0.005
0.016
I.20
3.5
0.66
1.9
Epabestat
-
0.012
-
2.6
-
0.75
-
Tolrestat
0.020
IC,
values were estimated from the least squares regression line of the log dose-response plot. HT, human testis; HE, human erythrocytc; RT, rat testis; RL, rat lens; RK, rat kidney. ‘Data from Nakayama et al. (1989). “Data from Kawasaki er al. (1989). Data from Sato el 01. (1988).
exhibited a biphasic kinetics pattern with pr-giyceraldehyde (K,,, = 0.029 and 1.5 mM) (Pig. 5). The effect of activator and inhibitors on PLP-treated aldose reductase was studied (Table 5). The activity of aldose reductase treated with PLP was unchanged in the presence of 0.3 M a~onium sulfate, though the
activity of native enzyme was increased cu 3 times with same concentration of ammonium sulfate. When aldose reductase was treated with PLP, the enzyme activity was protected from inhibition by aldose reductase inhibitor. The native enzyme was inhibited to 27 and 18% activity by 5 x lo-* M AL1 576 and
Human
testis aldose
and aldehyde
427
reductase
1/[DL-Glyceraldehyde]
(mM)
The enzyme (I x 10m6M) was treated with (+) or without (a) 250 PM PLP for 10 min. The enzyme activities were assayed as described in text except that various concentrations of rn-glyceraldehyde were added. Fig. 5. Lineweaver-Burk
plots for native and pyridoxal5’-phosphate
Table 5. Effect of activator and inhibitors on native and PLP treated-aldose reductase Relative activity (%) Activator or inhibitor None (NH,)2 80, Epalrestat AL1576
Cont. (M)
Native
0.3 2 X 10-s 5 X 10-7 5 X 10-s 2.5 X 10-s
100 318 5.7 17.8 26.9 41.9
PLP treated 268 242 114 198 252 265
(100) (90.4) (42.7) (73.7) (93.9) (98.9)
The enzyme (I x IO-‘M) was incubated with or without 0.5mM PLP in 50 mM phosphate buffer, pH 7.0, at 25°C. The activity of the enzyme incubated for 10 min was assayed in the presence of the indicated concentration of activator or inhibitor according to the method described in the text. The enzyme activities were expressed as percentage with respect to the activity of native enzyme with no activator and inhibitor. The numbers in parentheses were percentages with respect to PLP treated-enzyme activity without activator and inhibitor. Native, enzyme “ntreated with PLP; PLP treated, enzyme treated with PLP. 5 x lo-‘M Epalrestat, respectively. On the other hand, PLP-treated enzyme was not almost inhibited by these inhibitors. It was speculated from this finding that aldose reductase inhibitor interacted with lysine residue in aldose reductase from human testis. SUMMARY
Aldose reductase (alditol : NADP+ l-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol: NADP+ oxidoreductase, EC 1.1.1.2) were purified to homogeneity from human testis. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as D-xylose, o-glucose and o-galactose, whereas aldehyde reductase was virtually inactive for aldosugars. The K,,, values of aldose reductase for D-ghcase, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. Sulfate ion caused
treated aldose reductase.
3-fold activation of aldose reductase, but little for that of aldehyde reductase. Sodium valproate inhibited significantly aldehyde reductase, but aldose reductase was inhibited only very slightly by valproate. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10-7-10-9 M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. It can be presumed that the administration of aldose reductase inhibitors to patients with diabetes, may affect the system of male reproduction. Reaction of aldose reductase with PLP resulted ca 2.5-fold activation, but aldehyde reductase reacted with PLP did not cause the activation. PLP-treated aldose reductase was not activated by ammonium sulfate, and has lost the susceptibility to aldose reductase inhibitors. From this finding, it was suggested that aldose reductase inhibitor interacts with a lysine residue in aldose reductase molecule. REFERENCES
Boghosian R. A. and McGuinness E. T. (1979) Affinity purification and properties of porcine brain aldose reductase. Biochim. biophys. Acta 567, 278-286. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-binding. Analyf. Biochem. 72, 248-254.
Buszney S. M., Frank R. N., Verma S. D., Tanishima T. and Gabbay K. H. (1977) Aldose reductase in retinal mural cells. Invest. Ophthalmol. Vis. Sci. 16, 392-396. Cleland W. W. (1963) Computer programmes for processing enzyme kinetic data. Nature 198, 46345. Clements R. S. Jr and Winegrad A. I. (1972) Purification of alditol: NADP oxidoreductase from human placenta. Biochem. biophys. Res. Commun. 47, 1413-1479.
Cromlish J. A. and Flynn T. G. (1983a) Purification and characterization of two aldose reductase isoenzymes from rabbit muscle. J. biol. Chem. 2SIl, 34163424. Cromlish J. A. and Flynn T. G. (1983b) Pig muscle aldehyde reductase: identity of pig muscle aldehyde reductase with pig lens aldose reductase and with the low K,,, aldehyde reductase of pig brain and pig kidney. J. biol. Chem. 258, 3583-3586.
428
Tswos~t TANIMOKI et al.
Das B. and Srivastava S. K. (1985) Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte. Archs Biochem. Biophys. 238, 670-679.
Gabbay K. H. (1975) Hyperglycemia, polyol metabolism and complications of diabetes mellitus. A. Rev. Med. 26, 521-536.
Haymann S. and Kinoshita J. H. (1965) Isolation and properties of lens aldose reductase. J. biol. Chem. 240, 877-882.
Herrmann R. K., Kador P. F. and Kinoshita J. H. (1983) Rat lens aldose reductase: rapid purification and comparison with human placental aldose reductase. Exp. Eye Res. 31, 467474.
Hers H. G. (1960) L’aldose-reductase.
Biochim. biophys.
Acta 37, 120-126.
Inagaki K., Miwa I. and Okuda J. (1982) Affinity purification and glucose specificity of aldose reductase from bovine lens. Archs Biochem. Biophys. 216, 337-344. Jaspan J., Maselli R., Herold K. and Bartkus C. (1983) Treatment of severely painful diabetic neuropathy with an aldose reductase inhibitor: relief of pain and improved somatic and autonomic nerve function. Lancer Oct. 1, 758-762.
Jedzewitsch R. G., Jaspan J. B., Polonsky K. S., Weinberg C. R., Halter J. B., Halar E., Pfeifer M. A., Vukadinovic C., Bernstein A. H., Schneider M., Liang K-Y., Gabbay K. H.. Rubenstein A. H. and Porte D. Jr (1983) Aldose reductase inhibition improves nerve conduction~ velocity in diabetic patients. New Engl. J. Med. 308, 119-125. Kador P. F. and Kinoshita J. H. (1985) Role of aldose reductase in the development of diabetes-associated complications. Am. J. Med. 79, 8-12. Kador P. F., Merola L. D. and Kinoshita J. H. (1979) Differences in the susceptibility of various aldose reductase to inhibition. Docum. Ophrhal. Proc. Series 18, 117-124. Kawasaki N., Tanimoto T. and Tanaka A. (1989) Characterization of aldose reductase and aldehyde reductase from rat testis. Biochim. biophys. Acta 996, 30-36. Kern T. S. and Engerman R. L. (1982) Immunohistochemical distribution of aldose reductase. Histochem. J. 14, 507-515.
J. H. (1988) Polyol Pathway and its Role in Diabetic Complications, pp. 12-19. Elsevier, Amsterdam.
Kinoshita
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Ludvigson M. A. and Sorenson R. L. (1980) Immunohisto-
chemical localization of aldose reductase I. Enzyme purification and antibody preparation-localization in peripheral nerve, artery and testis. Diabetes 29, 43849. Markus H. B., Raducha M. and Harris H. (1983) Tissue distribution of mammalian aldose reductase and related enzymes. Biochem. Med. 29, 31-45. Moonsammy G. I. and Stewart M. A. (1967) Purification and properties of brain aldose reductase and L-hexonate dehydrogenase. J. Neurochem. 14, 1187-l 193. Morjana N. A. and Flynn T. G. (1989) Aldose reductase from human psoas muscle. J. biol. Chem. 264,2909-29 I 1. Morjana N. A., Lyons C. and Flynn G. T. (1989) Aldose reductase from human psoas muscle: affinity labeling of an active site lysine by pyridoxal 5’-phosphate and pyridoxal 5’-diphospho-5’-adenosine. J. biol. Chem. 264, 2912-2919. Nakayama T., Tanimoto T. and Kador P. F. (1989) Human erythrocyte aldose and aldehyde reductase. Prog. C/in. Biol. Res. 290, 265-277.
O’Brien M. M. and Schofield P. J. (1980) Polyol-pathway enzymes of human brain: partial purification and properties of aldose reductase and hexonate dehydrogenase. Biochem. J. 187, 21-30.
Okumura K., Lee P. U. and Dixon R. L. (1975) Permeability of selected drugs and chemicals across the blood-testis barrier of the rat. J. Pharm. Exp. Ther. 194, 89-95. Sato S., Kador P. F. and Kinoshita J. H. (1988) Polyol pathway and its Role in Diabetic Complications, pp. 12-19. Elsevier, Amsterdam. SheafIC. M. and Doughty C. C. (1976) Physical and kinetic properties of homogeneous bovine lens aldose reductase. J. biol. Chem. 251, 26962702. Srivastava S. K., Ansari N. A., Hair G. A. and Das B. (1984) Aldose and aldehyde reductases in human tissues. Biochim. biophys. Acra 800, 220-227.
Tanimoto T., Fukuda H. and Kawamura J. (1984) Characterization of aldose reductase Ia and Ib from rabbit lens. Chem. Pharm. Bull. 32, 1025-1031.
Tanimoto T., Fukuda H., Yamaha T. and Tanaka C. (1986) Reaction and inhibition mechanisms of aldose reductase from rabbit lens. Chem. Pharm. BUN. 34, 41834189. Tanimoto T., Sato S. and Kador P. F. (1990) Purification and properties of aldose reductase and aldehyde reductase from EHS tumor cells. Biochem. Pharmac. 39, 445453. Whittle S. R. and Turner A. J. (1981) Biogenic aldehyde metabolism in rat brain: differential sensitivity of aldehyde reductase isoenzymes to sodium valproate. Biochim. biophys. Acta 657, 94105.
Inf.J. Biochem.Vol. 23, No. 4, pp. 421-428, 1991 Printed in Great Britain. All rights reserved
PURIFICATION AND CHARACTERIZATION OF HUMAN TESTIS ALDOSE AND ALDEHYDE REDUCTASE TSUYOSHITANIMOTO,’MIYAKOOHTA,’AKIRATANAKA,’ISAOIKEMOTO’and TOYOHEIMACHIDA* ‘Division of Biological Chemistry, National Institute of Hygienic Sciences, l-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan [Tel. 700-I 1411 *Department of Urology, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan (Rem-bed 6 June l?WU)
Abstract-I.
Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pl values of these enzymes were found to be 5.9 and 5.1 by chromatof~using, respectively. 3. Aidose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The K,,, values of aldose reductase for o-glucose, D-galactose and D-XylOSe were 57, 49 and 6.2mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 1O-7-1O-g M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5’-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.
INTRODWCTION Aldose reductase (alditol : NADP+ I-oxidoreductase, EC 1.1.1.2 1) catalyzes the first reaction of the polyol pathway, and the enzyme catalyzes the conversion of aldose to the corresponding sugar-alcohol. Aldose reductase has been found to be present in various mammalian tissues (Gabbay, 1975; Kern and Engerman, 1982; Markus et al., 1983; Srivastava et al., 1984). However, its metabolic significance has only been studied in the seminal vesicles. Ludvigson and Sorenson (1980) demonstrated the presence of aldose reductase in rat testes using immunohist~hemi~l techniques. It has been suggested that testis aldose reductase together with sorbitol dehydrogenase constitutes the polyol pathway and may be responsible for the formation of D-fructose which has a trophic role for spermatides in late stages of s~~atogenesis. In recent years, interest in this enzyme has increased because of its possible role in the pathogenesis of diabetic complications such as cataract, retinopa~y, neuropathy and nephropathy (Buszney et al., 1977; Kador et al., 1979; Kador et al., 1985; Kinoshita, 1988). Indeed, some aldose reductase inhibitors have been found to be useful for preventing or treating chronic complications due to diabetes (Jed~wi~h et al., 1983; Jaspan et al., 1983). However, there are no studies with regard to the effect of these inhibitors on aldose reductase in tissues which are not directly associated with diabetic complications. A knowledge of the enzymatic properties and susceptibility to inhibition of aldose reductase in
tissues unaccompanied by diabetic complications would be useful for the elucidation of the side effects of aldose reductase inhibitors. In this paper, we report for the first time the purification, characterization and susceptibility to inhibition by commercially developed aldose reductase inhibitors of human testis aldose reductase and aldehyde reductase. MATERIALSAND METHODS Materials DL-Gly~raldehyde and rr-glucuronic acid were purchased from Aidrich Chemical Co: and the other aldbses from Wako Pure Chemical Industries Ltd. NADPH and NADH were obtained from Oriental Yeast Co. Sephadex G-75, Polybuffer 74 and Polybuffer exchanger 94 were purchased from Pharmacia Fine Chemicals. Molecular weight standard protein was obtained from Bio-Rad Laboratories. Matrex gel orange A was purchased from Amicon Co. Sorbinil [(S)-6-fluorospiro-(chroman-4,5’-imidazolidme)2’,4’-dione], tolrestat (N-[(S-tri-fluoro-methyl-6-methoxyI-naphthaIenyi)thioxomethyl]-~-methyl-~ycine), M79175 [2-methyl-6-fluorospiro-(chroman-4,5’-imidazolidine-~,~dione)], ponalrestat ([3-(4-bromo-2-fluorobenzyl)-4-oxo3H-phthalazin-l-yl]-acetic acid) and AL1576 (2,7-difluorospirofluorene-9,5’-imidazolidine-2’,4’-dione) were kindly donated by Dr Kador, National Institutes of Health. U.S.A. Epalrestat ((E,E)-5-[(2-methyl-3-phenyi-2-pro~nyiidene)4-oxo-2-thioxo-3-thiazolidinel-acetic acid) was a gift of Professor Tanaka, Osaka University of Pharmaceutical Sciences. The protein assay kit was obtained from Bio-Rad Laboratories. Testes were obtained from human and stored frozen at -80°C.
421
422
Tsuvowr
TANIMOKJ et al.
Table 1. Purification of aldose reductase and aldehvde reductase from human testis
Step Extraction Matrex gel orange A Sephadex G-75 Chromatofocusing Aldose reductase (peak I) Aldehyde reductase (peak II)
Total protein (mg)
Total activity (U)
Sp. act. (Wmg)
2886 8.32 5.85
4.65 3.51 3.41
0.0016 0.429 OS83
0.916 1.030
1.32 2.66
0.694 0.387
Assay of enzyme activity
The activities of aldose reductase and aldehyde reductase were determined at 25°C by measuring the decrease in absorption of NADPH at 340nm on a Hitachi U-2090 spectrophotometer equipped with a temperature-controlled cuvette chamber. Assay mixture in a l.Oml system contained 90 mM sodium phosphate buffer (pH 6.2), 0.15 mM NADPH and 10mM m-glyceraldehyde. The reaction was initiated by adding the enzyme, and the rate of NADPH oxidation was followed by recording the decrease in absorbance at 340 nm. The appropriate blank for correction of nonspecific oxidation of NADPH was prepared. One unit of the enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 pmol of NADPH per min under the conditions described here. Determination
of kinetic constant
initial rate determination of each substrate was carried out in duplicate at six concentrations, and the values were calculated with a computer using the programs of Cleland (1963). The
Determination
of IC,
The concentration of inhibitor giving 50% inhibition of the enzyme activity (IC,) was estimated from the least squares regression line of the log dose-response plot. Protein
&termination
concentration of protein was determined with the Bio-Rad protein assay kit according to the method of Bradford (1976). The
Polyacrylamide
gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on slab gels containing a 12% acrylamide according to the method of Laemmli (1970). Protein were visualized with Coomassie Brilliant Blue R250. Standard proteins used for estimation of molecular weight were trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa). Enzyme purification
All operations were performed at 0-4”C. All buffers contained 2mM dithiothreitol in order to protect the enzyme from inactivation. Step 1. Extraction: human testis of 12 g was homogenized in 30 ml of 20 mM sodium phosphate buffer @H 7.0) with Polytron, and the homogenate was centrifuged at 15,OOOg for 30 min to remove insoluble materials. Step 2. Matrex gel orange A afinity chromatography: the extract of human testis was diluted to 3-times with 20 mM phosphate buffer (pH 7.0), and was applied directly to a column (1.5 x 25 cm) of Matrex gel orange A equilibrated with 20 mM sodium phosphate buffer @H 7.0). After the column was thoroughly washed with the same buffer, the enzyme was eluted with a linear concentration gradient of sodium chloride, formed from lSOm1 of 20mM sodium phosphate buffer @H 7.0) in the mixing chamber and 150 ml of the same buffer containing 0.6 M sodium chloride in the
Purification (fold)
I
Yield W)
267 364
100 75.5 73.3
825 I663
19.7 22.2
reservoir, at a flow rate of 25 ml per hr. The enzyme activity was observed in the fraction eluted with ca 0.25 M sodium chloride. Fractions with enzyme activities were pooled and concentrated by ultrafiltration using a collodion bag. Step 3. Sephadex G- 7.5 gel jiltration: the concentrated enzyme solution from Step 2 was placed on a columm (1.2 x 50 cm) of Sephadex G-75 equilibrated with 25 mM imidazole_HCl buffer (pH 7.2) and was eluted with the same buffer at a flow rate of 10ml per hr. The enzyme fractions obtained were pooled and concentrated by ultrafiltration using a collodion bag. Step 4. Chromatofocusing: the concentrated enzymes solution described above was sub&ted to chromatofocusinn on a column (1 x 40 cm) of polybuffer exchanger 94 equilc brated with 25 mM imidazoleHC1 buffer (OH 7.2). The elution was carried out with 1:8 diluted polybufi’er 74 (PH 4.5) at a flow rate of 20 ml per hr, and fractions of 2.2 ml were collected. RESULTS AND DISCUSSION Enzyme purification Two NADPH-dependent reductases have been purified from human testis in the four steps summar-
ized in Table 1. With DL-glyceraldehyde as substrate only a single peak of NADPH-dependent reductase activity was obtained from affinity chromatography on Matrex gel orange A and gel filtration on Sephadex G-75. Subsequent chromatofocusing of enzyme fractions obtained from gel filtration resulted in the appearance of two peaks of enzyme activity, tentatively designated as peaks I and II (Fig. 1). On the basis of enzymatic properties of these enzymes described in this paper, the enzymes in peaks I and II were identified as aldose reductase and aldehyde reductase, respectively. Aldose reductase in human testis was purified ca 820-fold with the sp. act. of 1.32 U per mg, and was recovered in ca 20%. Aldehyde reductase was purified ca 1660-fold with a sp. act. of 2.66 U per mg, and the yield was ca 22%. Molecular weights and isoelectric points The molecular weights of human testis aldose reductase and aldehyde reductase were estimated to IX 36,000 and 38,000 by SDS-PAGE respectively
(Fig. 2). The molecular weights of human testis aldose reductase and aldehyde reductase were similar to those reported for both enzymes from various other sources (Clements and Winegrad, 1972; Sheaff and Doughty, 1976; Boghosian and McGuinness, 1979; Herrmann et al., 1983). The p1 values determined by chromatofocusing were 5.9 for aldose reductase and 5.1 for aldehyde reductase. In mammalian tissues such as ox brain (Moonsammy and Stewart, 1967), rat testis (Kawasaki et al., 1989) and EHS tumor cell (Tanimoto et al., 1990), aldose reductase was more acidic protein than aldehyde
423
Human testis aldose and atdehyde reductase
Fraction number
Fig.
1. ~hr~matof~usj~g
of aldose reductase and aldehyde reductase from human testis. The pooled
fractions obtained from gel filtration was applied to a c~romatof~~ing column. Fractions of i Ximl were measured for protein content (---), enzyme activity (a) and pH c.-). reductase. However, the p1 value of aldose reductase in human tissues was higher than that of aldehyde reductase (O’Brien and Schofield, 1980, Srivastava et al., 1984; Das et al., 1985).
The kinetic constants of aldose reductase and aldehyde reductase were determined for several substrates and are given in Table 2. Aldose reductase reduced aldo-sugars such as o-xylose, ~-glucose and D-g$aCtOSe, and the Km values for these substrates were 6.2, 57, 49mM, respectively. Compared to Dt-glyceraldehyde, aldose reductase displayed relatively high K,,, values for D-xylose, D-glucose and D-galactose and significantly low k,,/K, values for these aldo-sugars. However, because the aldehyde form of aldo-sugars is the real substrate for aldose reductase, as pointed out by Inagaki et al. (1932), accounting for the aldehyde form of a-glucose and n-galactose leads to very low corrected K, values (6.9 BM for D-glucose and 34 FM for D-galactose) and high k,,jii, values (89,~O for o-glucose and 20,500 for D-galactose). The apparent X, values for various aldo-sugars of human testis aldose reductase were lower than those of human psoas muscle enzyme (Morjana and Flynn, 1989), and were in the same range as those reported for the enzyme isolated from rabbit muscle (Cromlish and Flynn, 1983a), human erythrocyte (Das et al., 1985), human placenta (Clements and Winegard, 1972) and rat lens Table 2. Substrate
(Herrmann et al., 1983). Aldose reductase can also reduce D-glucuronate as substrate, but this enzyme displayed a Km value 200-times greater and a k,, JX;, value lo-tirn~ less than those for DL-gly~eraidehyde. The &, value of aldehyde reductase for DL-glyceraldehyde was found to be higher than that of aldose reductase. Aldehyde reductase was virtually inactive for aldo-sugars. The Km vafue of aldehyde reductase for D-gfucuronate was similar to the value for Dt-glyceraldehyde, and the k-*/k;, value for D-glucuronate was 1.5 times higher than DL-gly~raldehyde‘ With respect to coenzyme specificity, aldose reductase utilized both NADPH and NADH as coenzymes. However, the enzyme showed a preference for NADPH. This is indicated by the low K,,, value for NADPH (3.2 PM) as compared to that for NADH (250 ~JM), On the other hand, aldehyde reductase exhibited no enzyme activity with NADH as a eofaetor, and utilized only NADPH as a coenzyme, the K,,, being 3.8 /.iM.
The effects of salts on aldose reductase and aldehyde reductase activities are shown in Table 3. The activity of aldose reductase was increased 3-4times in the presence of 0.3 M ammonium sulfate. Similar activation was also observed in the presence of 0.3 M sodium sulfate. However, 0.3 M ammonium chloride and sodium chloride slightly inactivated aldose reductase. Sodium chloride or ammonium chloride
specificities and kinetic constants of human testis aldose reductase and aldehyde reductase
Aldose reductase
AIdehyde reductase
k
xx.-Giyceraldehyde D-xyiOS.5 D-Giuccse
D-Galactose a-Glucuronate NADPH NADH
k
(=?f
Substrate t3.026 f. 0.001 6.2 + 0.43 57.3 + 3.3 49.3 * 1.7 5.05 rt 0.21 0.0032 rf 0.0003 0.25 * 0.01
0.672 0.633 0.614 0.702 0.697
* F f + +
&&n 0.026 0.043 0.035 0.024 0.029
25,850 102 11 14 138 -
3.17 kO.39 >3ow >45oO >4500 4.69 f 0.27 0.0038 & 0.0004 ND
(se‘?!)
&t/K”
1.33_tO.16 2.34t_O.13 -
420 499 -
For the determination of K, values for different substrates, the concentration of NADPH was kept at 0.15 mM. For the determination of K, values for NAD(P)H, DL-gtyceraldehyde concentration was kept at 3 mM for aidose reductase and 30 mM for aldehyde reductase. ND, not detectable.
94K
-
ZOK
-
Fig. 2. SDS-PAGE of the purified aldosc reductase and aldehyde rcductase from human testis. The gel was stained with Coomassie Brilliant Blue R250. Lane S, standard proteins; lane 1, aldose reductase; lane 2, aldehyde reductase.
424
425
Human testis aldose and aldehyde reductase Table 3. Effect of salts on human testis aldose reductase and aldehyde reductase Relative activity (%)
Salt
Aldose reductase
Aldehyde reductase
None
100
100
(NW=4
338 296 84 98
91 98 76 74
Na,SO, NaCl NH,CI
Effects of different salts on the activities of aldose reductase and aldehyde reductase were studied at 0.3 M concentration.
could not be substituted for sodium sulfate or ammonium sulfate, indicating that sulfate ion was the activating species for aldose reductase. The response of aldose reductase to sulfate ion was similar to that of rat (Herrmann et al., 1983), calf (Haymann and Kinoshita, 1965) and rabbit (Tanimoto et al., 1984) lens, sheep seminal vesicle (Hers, 1960), human erythrocyte (Das et af., 1985) and placenta (Clements and Winegrad, 1972) aldose reductase. In contrast to aldose reductase, aldehyde reductase activity was not affected by 0.3 M ammonium sulfate and sodium sulfate. The enzyme, however, was inactivated slightly by 0.3 M ammonium chloride and sodium chloride as well as aldose reductase. Effect of various inhibitors Valproate, which selectively inhibits aldehyde reductase (Whittle and Terner, 1981; Cromlish and Flynn, 1983b) was a much better inhibitor of human testis aldehyde reductase than of aldose reductase. Sodium valproate at 0.5 mM inhibited aldehyde reductase by ca 90%, but aldose reductase was inhibited only very slightly by the same concentration (Fig. 3). The susceptibility of human testis aldose reductase and aldehyde reductase to inhibition by the commercially developed aldose reductase inhibitors such as sorbinil, tolrestat, M79175, ponalrestat, AL1576 and epalrestat, is summarized in Table 4. Human testis aldose reductase was greatly inhibited by these inhibitors at concentrations of the order of 10-7-10-9 M. Inhibition of human testis aldose reductase appeared to be similar to that observed with aldose reductase from rat testis (Kawasaki et al., 1989), and human
testis aldose reductase was inhibited to a greater extent than the enzyme from human erythrocyte (Nakayama et al., 1989). On the other hand, all six compounds were found to inhibit human testis aldehyde reductase appears to be less susceptible to these inhibitors than aldose reductase. The susceptibility to inhibition of human testis aldehyde reductase was almost the same as that of human erythrocyte (Nakayama et al., 1989), rat testis (Kawasaki et al., 1989) and rat kidney (Sato et al., 1988) enzymes. Aldose reductase together with sorbitol dehydrogenase forms a polyol pathway, and by this pathway D-fructose which is a life energy source to spermatozoa is synthesized. It was reported that aldose reductase was localized to the Sertoli cell and mature spermatids in rat testis (Ludvigson and Sorenson, 1980). Therefore, aldose reductase in the testis may play an important trophic role in spermatids in the late stages of spermatogenesis. Although the permeability of drugs from blood to germ ceil was regulated by the blood-testis barrier (Okumura et al., 1975) and it is not yet clear whether administered aldose reductase inhibitor distribute in a germ cell, it can be presumed from the results described in this paper that the administration of aldose reductase inhibitors, which have been developed for the prevention or treatment of chronic complications due to diabetes, may affect the system of human male reproduction. There may be no gain in saying that aldose reductase inhibitors produce undesirable side-effects in clinical use. Effect of pyridoxal S-phosphate on aldose reductase The reaction of aldose reductase from human testis with 250 and 500 PM of pyridoxal 5’-phosphate (PLP) resulted ca 2 to 2.5-fold activation of the enzyme, however, PLP did not affect on aldehyde reductase (Fig. 4). The activation by PLP was also observed for human psoas muscle aldose reductase (Morjana et al., 1989), but not observed for rat testis (Kawasaki et al., 1989) and rabbit lens (Tanimoto et al., 1986) aldose reductase. Therefore, it may be specific for human aldose reductase that the enzyme is activated by PLP. Although native aldose reductase exhibited monophasic kinetics with DL-glyceraldehyde (K,,, = 0.026 mM), the PLP-activated enzyme
500,
60 Time (min)
Sodium valproate
(mM)
Fig. 3. Inhibition of human testis aldose reductase (0) and aldehyde reductase (m) by sodium valporate. The enzyme activities were assayed in the presence of the indicated concentration of sodium valporate.
Fig. 4. Effect of PLP on human testis aldose reductase and aldehyde reductase. The enzyme (1 x 10e6M) was incubated at 25°C with PLP at the following concentrations: 500 p M (0) and 250 p M (A) for aldose reductase and
500 PM (W) for aldehyde reductase. Aliquots were removed at the various times and were assayed for enzyme activities.
426
TSUKISHITANIMOTO et
of.
Table 4. Effeot of aldose reductase inhibitors on human testis aldose reductase and aldehyde redwtase IC,
tiM)
Aldose reductase
Aldehyde reductase
Inhibitor
HT
HF
RF
RL’
Sotbinil
0.55
1.8
0.18
0.07
M79175
0.044
1.4
0.053
0.055
0.017
AL1576
HT
HE”
RTb
RK’
4.6
7.6
1.5
1.8
0.028
0.16
0.71
0.10
0.16
0.024
0.01 I
0.025
0.012
0.046
1.2
0.50
0.24
0.013
0.010
0.011
0.75
Ponahestat
0.13
0.005
0.016
I.20
3.5
0.66
1.9
Epabestat
-
0.012
-
2.6
-
0.75
-
Tolrestat
0.020
IC,
values were estimated from the least squares regression line of the log dose-response plot. HT, human testis; HE, human erythrocytc; RT, rat testis; RL, rat lens; RK, rat kidney. ‘Data from Nakayama et al. (1989). “Data from Kawasaki er al. (1989). Data from Sato el 01. (1988).
exhibited a biphasic kinetics pattern with pr-giyceraldehyde (K,,, = 0.029 and 1.5 mM) (Pig. 5). The effect of activator and inhibitors on PLP-treated aldose reductase was studied (Table 5). The activity of aldose reductase treated with PLP was unchanged in the presence of 0.3 M a~onium sulfate, though the
activity of native enzyme was increased cu 3 times with same concentration of ammonium sulfate. When aldose reductase was treated with PLP, the enzyme activity was protected from inhibition by aldose reductase inhibitor. The native enzyme was inhibited to 27 and 18% activity by 5 x lo-* M AL1 576 and
Human
testis aldose
and aldehyde
427
reductase
1/[DL-Glyceraldehyde]
(mM)
The enzyme (I x 10m6M) was treated with (+) or without (a) 250 PM PLP for 10 min. The enzyme activities were assayed as described in text except that various concentrations of rn-glyceraldehyde were added. Fig. 5. Lineweaver-Burk
plots for native and pyridoxal5’-phosphate
Table 5. Effect of activator and inhibitors on native and PLP treated-aldose reductase Relative activity (%) Activator or inhibitor None (NH,)2 80, Epalrestat AL1576
Cont. (M)
Native
0.3 2 X 10-s 5 X 10-7 5 X 10-s 2.5 X 10-s
100 318 5.7 17.8 26.9 41.9
PLP treated 268 242 114 198 252 265
(100) (90.4) (42.7) (73.7) (93.9) (98.9)
The enzyme (I x IO-‘M) was incubated with or without 0.5mM PLP in 50 mM phosphate buffer, pH 7.0, at 25°C. The activity of the enzyme incubated for 10 min was assayed in the presence of the indicated concentration of activator or inhibitor according to the method described in the text. The enzyme activities were expressed as percentage with respect to the activity of native enzyme with no activator and inhibitor. The numbers in parentheses were percentages with respect to PLP treated-enzyme activity without activator and inhibitor. Native, enzyme “ntreated with PLP; PLP treated, enzyme treated with PLP. 5 x lo-‘M Epalrestat, respectively. On the other hand, PLP-treated enzyme was not almost inhibited by these inhibitors. It was speculated from this finding that aldose reductase inhibitor interacted with lysine residue in aldose reductase from human testis. SUMMARY
Aldose reductase (alditol : NADP+ l-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol: NADP+ oxidoreductase, EC 1.1.1.2) were purified to homogeneity from human testis. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as D-xylose, o-glucose and o-galactose, whereas aldehyde reductase was virtually inactive for aldosugars. The K,,, values of aldose reductase for D-ghcase, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. Sulfate ion caused
treated aldose reductase.
3-fold activation of aldose reductase, but little for that of aldehyde reductase. Sodium valproate inhibited significantly aldehyde reductase, but aldose reductase was inhibited only very slightly by valproate. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10-7-10-9 M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. It can be presumed that the administration of aldose reductase inhibitors to patients with diabetes, may affect the system of male reproduction. Reaction of aldose reductase with PLP resulted ca 2.5-fold activation, but aldehyde reductase reacted with PLP did not cause the activation. PLP-treated aldose reductase was not activated by ammonium sulfate, and has lost the susceptibility to aldose reductase inhibitors. From this finding, it was suggested that aldose reductase inhibitor interacts with a lysine residue in aldose reductase molecule. REFERENCES
Boghosian R. A. and McGuinness E. T. (1979) Affinity purification and properties of porcine brain aldose reductase. Biochim. biophys. Acta 567, 278-286. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-binding. Analyf. Biochem. 72, 248-254.
Buszney S. M., Frank R. N., Verma S. D., Tanishima T. and Gabbay K. H. (1977) Aldose reductase in retinal mural cells. Invest. Ophthalmol. Vis. Sci. 16, 392-396. Cleland W. W. (1963) Computer programmes for processing enzyme kinetic data. Nature 198, 46345. Clements R. S. Jr and Winegrad A. I. (1972) Purification of alditol: NADP oxidoreductase from human placenta. Biochem. biophys. Res. Commun. 47, 1413-1479.
Cromlish J. A. and Flynn T. G. (1983a) Purification and characterization of two aldose reductase isoenzymes from rabbit muscle. J. biol. Chem. 2SIl, 34163424. Cromlish J. A. and Flynn T. G. (1983b) Pig muscle aldehyde reductase: identity of pig muscle aldehyde reductase with pig lens aldose reductase and with the low K,,, aldehyde reductase of pig brain and pig kidney. J. biol. Chem. 258, 3583-3586.
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Tswos~t TANIMOKI et al.
Das B. and Srivastava S. K. (1985) Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte. Archs Biochem. Biophys. 238, 670-679.
Gabbay K. H. (1975) Hyperglycemia, polyol metabolism and complications of diabetes mellitus. A. Rev. Med. 26, 521-536.
Haymann S. and Kinoshita J. H. (1965) Isolation and properties of lens aldose reductase. J. biol. Chem. 240, 877-882.
Herrmann R. K., Kador P. F. and Kinoshita J. H. (1983) Rat lens aldose reductase: rapid purification and comparison with human placental aldose reductase. Exp. Eye Res. 31, 467474.
Hers H. G. (1960) L’aldose-reductase.
Biochim. biophys.
Acta 37, 120-126.
Inagaki K., Miwa I. and Okuda J. (1982) Affinity purification and glucose specificity of aldose reductase from bovine lens. Archs Biochem. Biophys. 216, 337-344. Jaspan J., Maselli R., Herold K. and Bartkus C. (1983) Treatment of severely painful diabetic neuropathy with an aldose reductase inhibitor: relief of pain and improved somatic and autonomic nerve function. Lancer Oct. 1, 758-762.
Jedzewitsch R. G., Jaspan J. B., Polonsky K. S., Weinberg C. R., Halter J. B., Halar E., Pfeifer M. A., Vukadinovic C., Bernstein A. H., Schneider M., Liang K-Y., Gabbay K. H.. Rubenstein A. H. and Porte D. Jr (1983) Aldose reductase inhibition improves nerve conduction~ velocity in diabetic patients. New Engl. J. Med. 308, 119-125. Kador P. F. and Kinoshita J. H. (1985) Role of aldose reductase in the development of diabetes-associated complications. Am. J. Med. 79, 8-12. Kador P. F., Merola L. D. and Kinoshita J. H. (1979) Differences in the susceptibility of various aldose reductase to inhibition. Docum. Ophrhal. Proc. Series 18, 117-124. Kawasaki N., Tanimoto T. and Tanaka A. (1989) Characterization of aldose reductase and aldehyde reductase from rat testis. Biochim. biophys. Acta 996, 30-36. Kern T. S. and Engerman R. L. (1982) Immunohistochemical distribution of aldose reductase. Histochem. J. 14, 507-515.
J. H. (1988) Polyol Pathway and its Role in Diabetic Complications, pp. 12-19. Elsevier, Amsterdam.
Kinoshita
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Ludvigson M. A. and Sorenson R. L. (1980) Immunohisto-
chemical localization of aldose reductase I. Enzyme purification and antibody preparation-localization in peripheral nerve, artery and testis. Diabetes 29, 43849. Markus H. B., Raducha M. and Harris H. (1983) Tissue distribution of mammalian aldose reductase and related enzymes. Biochem. Med. 29, 31-45. Moonsammy G. I. and Stewart M. A. (1967) Purification and properties of brain aldose reductase and L-hexonate dehydrogenase. J. Neurochem. 14, 1187-l 193. Morjana N. A. and Flynn T. G. (1989) Aldose reductase from human psoas muscle. J. biol. Chem. 264,2909-29 I 1. Morjana N. A., Lyons C. and Flynn G. T. (1989) Aldose reductase from human psoas muscle: affinity labeling of an active site lysine by pyridoxal 5’-phosphate and pyridoxal 5’-diphospho-5’-adenosine. J. biol. Chem. 264, 2912-2919. Nakayama T., Tanimoto T. and Kador P. F. (1989) Human erythrocyte aldose and aldehyde reductase. Prog. C/in. Biol. Res. 290, 265-277.
O’Brien M. M. and Schofield P. J. (1980) Polyol-pathway enzymes of human brain: partial purification and properties of aldose reductase and hexonate dehydrogenase. Biochem. J. 187, 21-30.
Okumura K., Lee P. U. and Dixon R. L. (1975) Permeability of selected drugs and chemicals across the blood-testis barrier of the rat. J. Pharm. Exp. Ther. 194, 89-95. Sato S., Kador P. F. and Kinoshita J. H. (1988) Polyol pathway and its Role in Diabetic Complications, pp. 12-19. Elsevier, Amsterdam. SheafIC. M. and Doughty C. C. (1976) Physical and kinetic properties of homogeneous bovine lens aldose reductase. J. biol. Chem. 251, 26962702. Srivastava S. K., Ansari N. A., Hair G. A. and Das B. (1984) Aldose and aldehyde reductases in human tissues. Biochim. biophys. Acra 800, 220-227.
Tanimoto T., Fukuda H. and Kawamura J. (1984) Characterization of aldose reductase Ia and Ib from rabbit lens. Chem. Pharm. Bull. 32, 1025-1031.
Tanimoto T., Fukuda H., Yamaha T. and Tanaka C. (1986) Reaction and inhibition mechanisms of aldose reductase from rabbit lens. Chem. Pharm. BUN. 34, 41834189. Tanimoto T., Sato S. and Kador P. F. (1990) Purification and properties of aldose reductase and aldehyde reductase from EHS tumor cells. Biochem. Pharmac. 39, 445453. Whittle S. R. and Turner A. J. (1981) Biogenic aldehyde metabolism in rat brain: differential sensitivity of aldehyde reductase isoenzymes to sodium valproate. Biochim. biophys. Acta 657, 94105.
