Clinica Chimica Acta, 205 (1992) 109-116 © 1992 Elsevier Science Publishers B.V. All rights reserved 0009-8981192/$05.00
109
CCA 05213
Homocarnosinosis patients and great apes have a serum protein that cross-reacts with human serum carnosinase M e l C. J a c k s o n a n d J a m e s F. L e n n e y Pharmacology Department, School of Medicine, University of Hawaii, Honohdu, HI 96822 (USA) (Received 12 August 1991; revision received 30 October 1991; accepted 8 November 1991)
Key words: Serum carnosinase; Dipeptidase; Homocarnosinosis; Great apes; Antigen capture
Summary A specific polyclonal antiserum to human serum carnosinase was raised in rabbits and was used to prepare an agarose-protein A-antibody matrix. An antigen capture procedure showed that sera from homocarnosinosis patients, which lack carnosinase activity, contain an immunoreactive protein (M r 75 000) indistinguishable from the carnosinase band from normal serum. Other higher primates have active serum carnosinase and a similar immunoreactive Mr 75 000 protein. The immunoaffinity matrix was used in a facile procedure to isolate pure carnosinase from human plasma with a yield of 69%. The antiserum inhibited human serum earnosinase strongly, but the maximum inhibition attained averaged only 71%. The antiserum inhibited human and chimpanzee serum carnosinases more effectively than gorilla or other higher primate serum carnosinases.
Introduction Human serum carnosinase is a dipeptidase which is especially active in hydrolyzing carnosine (/~-alanylhistidine) and anserine (/3-alanyl-l-methylhistidine); it also splits homocarnosine (7-aminobutyric acid (GABA)-histidine) [1]. The active enzyme is a dimer, the two subunits (Mr 75 000) being connected by one or more Correspondence to: Mel C. Jackson, Pharmacology Department, School of Medicine, University of Hawaii, 1960 East West Road, Honolulu, H! 96822, U.S.A. Abbreviation: BBS, 0.1 mol/i sodium borate buffer (pH 8.2) containing 0.9% NaCI.
II0
disulfide bonds [1]. Serum carnosinase appears to be secreted by brain cells into cerebrospinal fluid, which transports it into the blood stream [I]. Carnosine hydrolyzing activity is present in higher primate sera, but when 12 non-primate mammals were tested, none exhibited serum activity except the Golden hamster [1]. Homocarnosinosis is a rare human metabolic disorder; Sjaastad et al. found that patients with this disease have cerebral spinal fluid (CSF) homocarnosine concentrations that are elevated 20-fold above normal [2]. These patients lack serum carnosinase activity [3] and this appears to explain the abnormal concentrations of CSF homocarnosine. We now report that the homocarnosinosis patients have an immunoreactive serum protein which appears to be an inactive form of carnosinase. Other higher primates and the Golden hamster also have a serum protein which cross-reacts with human serum carnosinase.
Experimental Materials Protein A-Sepharose 4B and all other chemicals were from Sigma Chemical Co.
Development of polyclonal antiserum Homogeneous human serum carnosinase was prepared from plasma as previously described [1]. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the enzyme band was excised and macerated with an equal volume of Freund's complete adjuvant. Approximately 35 ttg of enzyme was injected into a New Zealand white rabbit intradermally at multiple sites. Two weeks later the rabbit was reinjected with 35 #g ofantigen in the presence of Freund's incomplete adjuvant; this procedure was repeated after 4 weeks. Seven weeks after the initial injections the rabbit was exsanguinated via the femoral artery and the serum was stored in small aliquots at -70°C. Antibody titer was monitored during the immunization period by preincubating 50/zl of test-bleed serum with 150 #1 of a human serum carnosinase preparation for 30 min at 30°C. Aliquots (25 and 50/~i) were removed from the mixture and from control solutions containing enzyme without antiserum; these were assayed for activity against carnosine. Antibody titer was also measured using the enzyme-linked immunosorbent assay of Engvall and Perlmann [4] as modified by Chang et ai. [5].
Enzyme assays Serum carnosinase activity against carnosine and homocarnosine was measured using previously described procedures [6]. Free histidine liberated from the substrate was quantitated fluorometrically after reaction with o-phthaldialdehyde under conditions specific for histidine. Human tissue carnosinase activity against carnosine was measured under conditions optimal for this dipeptidase [7]. It is recommended that this enzyme, which is identical to prolinase, be called non-specific cytosolic
ill
dipeptidase [8]. The inhibitory activity of antiserum was measured after preincubation of enzyme with antiserum at 30°C as described above.
Double immunodiffusion Antibody monospecificity was tested using the double immunodiffusion procedure of Ouehterlony [9], in 1.5% agarose gels.
Preparation of immunoaffinity complex lmmunoaffinity chromatography matrix was prepared essentially as described by Schneider et al. [I0], incubating the antiserum with protein A-Sepharose 4B overnight at 4°C.
Immunoaffinity purification of human serum carnosinase Human plasma was chromatographed on a DEAE-cellulose column [11 and the fractions containing carnosinase were pooled and concentrated by ultrafiltration. Concentrate (15 ml) was mixed with 1 ml of Sepharose-protein A-antibody beads and 30 ml of BBS; the mixture was rotated for 24 h at 4°C. Elution of bound protein [I0] was confirmed by SDS-PAGE (4 mA, 16 h) followed by immersion of the gel in 4 mol/l sodium acetate [I 1]. The major protein band was excised, the gel slice was chopped into l-ram 3 cubes and the enzyme was electroeluted following the method of Hunkapiller and Lujan [12]; eluted enzyme was stored at -70°C.
Antigen capture and visualization Serum samples were assayed for immunoreactivity with serum carnosinase antibody by incubating 100 #1 of serum with 50 tzl of the Sepharose-protein A-antibody beads and 6 ml of BBS while rotating for 16 h at 4°C. After washing in BBS, 50 #i of a 1:1 ethylene glycol-BBS mixture were added to release bound antigen. Eluted antigen was examined by SDS-gel electrophoresis (200 V, 4°C) under reducing conditions in 8.75% polyacrylamide gels; protein bands were visualized using the Coomassie Blue G-250 staining technique of Neuhoffet al. [13]. Molecular weight was estimated using carbonic anhydrase, ovalburnin, bovine serum albumin, phosphorylase b, B-galactosidase and myosin as marker proteins.
Protein assay Protein concentrations were measured using the micro bicinchoninic acid method of Smith et al. [14], with bovine serum albumin as the reference protein. Results and Discussion
Antibody specificity and titer Double immunodiffusion assay &the antiserum showed a single precipitation line
112
with all antigen preparations tested. No spurs were seen at the junction of precipitation lines formed with crude or partially purified antigen preparations, indicating antibody monospeeificity. Enzyme-linked immunosorbent assay (ELISA) of the antiserum against purified serum carnosinase gave a titer of between 1:6250 and 1:31 250. Inhibition of serum carnosinase by antiserum
Figure 1 shows that human serum carnosinase was strongly inhibited by very low concentrations of antiserum. Incomplete inhibition at higher concentrations may be attributable to partial'blocking of the active center by antibodies or to a conformationai change affectin8 the active center. A similar maximum degree of inhibition
25
~
2O
0< .x-:. u.! ~ < zm ~03 O~ Z o
C~
~
lo
5
0
0
t 1
', 2
t 3
I 4
I 5
I 6
I 7
pl ANTISERUM / 0.5 ml DIGEST Fig. I. Effect of increasing amounts of antiserum on human serum carnosinase activity. Aliquots of a partially purified enzyme preparation (40 pl) were diluted to 200 #1 with 125 mmol/I NH4OH/HCI buffer (pH 8.5) containing 0-50 #l of antiserum. After 30 rain at 30*C, two aliquots from each tube were assayed for activity using carnosine as the substrate.
113 TABLE I Inhibition of higher primate serum carnosinases by antiserum to human serum carnosinase Serum source
Human Gorilla Chimpanzee Gibbon Pygmy Chimp
% Inhibition 30 rain
120 rain
42 6 45 0 0
56 22 49 4 0
Mixtures containing 50 pl serum, 100 ~tl 25 mmol/lN-ethylmorpholine/HCibuffer (pH 7.6) and 50 #1 antiserum were preincubated for 30 rain or 120 rain at 30°C. Control mixturescontaining no antiserum were also preincubated at 300C. Two 50-#1 aliquots from each tube were then assayedfor activityagainst carnosine.
was obtained when homocarnosine was employed as the substrate, suggesting that both dipeptides were hydrolyzed in the same active center. Table I shows the effect of the antiserum on the activity of serum carnosinases from five higher primates. The human and chimpanzee enzymes were inhibited to approximately the same extent, the gorilla enzyme was inhibited moderately, whereas little or no inhibition was seen with the gibbon and pygmy chimp enzymes. (Preimmune rabbit serum contained no carnosinase activity and had no inhibitory effect on human serum carnosinase). Evidently the structure o f the chimpanzee serum carnosinase resembles that of the human enzyme more closely than does the structure of the gorilla enzyme. Crude preparations of human kidney cytosolic non-specific dipeptidase [8] were inhibited by the antiserum in their hydrolysis of carnosine by an average of 9.5 ± 3.8% (n --- 4). This may be attributable to contamination of the preparations with serum carnosinase present in trapped blood. Golden hamster is the only non-primate animal known to have a serum carnosinase [1]. Surprisingly, the antiserum inhibited this enzyme 31 ± 4% (n = 4).
Immunoaffinity purification of serum carnosinase Immunoaffinity purification of human serum carnosinase, followed by preparative SDS-PAGE and electroelution from the gel, resulted in an apparently homogeneouS, protein. When this protein was analyzed by SDS-PAGE under reducing conditions, only one band was detected (Fig. 2). The M r of this protein was found to be 75 000 in agreement with the previously determined subunit Mr of human serum carnosinase purified by conventional methods [1]. By this procedure, approximately 550 #g of pure enzyme were obtained from 200 ml plasma, a 69% yield, assuming that plasma contains 4 mg enzyme per liter [ 1 ] .
114
~
..
d
@
.~_
:~
:!:,ii(
~
.~ -
=
f". .z.
._
U
U
~
~
,~
115
Antigen capture The antigen capture procedure was applied to serum samples from the three homocarnosinosis siblings, their mother and father and a normal control. Bound protein was eluted from the immunoaffinity matrix and subjected to SDS-PAGE. As shown in Fig. 3, all six eluates showed a single protein band at M r 75 000, indicating that the homocarnosinosis patients have an immunoreactive but enzymatically inactive form of serum earnosinase. Although the synthesis of this enzyme was defective, it was nonetheless secreted into the blood stream. Double immunodiffusion assay of these samples showed no spur formation when adjacent to normal human serum samples. Thus, the homocarnosinosis enzyme was antigenically identical to the normal enzyme with respect to this antiserum. When the antigen capture procedure was used with four higher primate serum samples, the results shown in Fig. 4 were obtained. A Mr 75 000" band was seen in all lanes, although the band was weak in the case of the pygmy chimp. Evidently these great ape serum carnosinases have the same Mr as the human enzyme and they also cross-react with it. Again, the Ouchterlony assay indicated that the great ape enzymes were antigenicaily the same as the normal human serum carnosinase. Serum from the orangutan showed a faint band at M r 70 000 and Golden hamster serum gave a faint band at Mr 65 000 (not shown). Rat serum, which does not hydrolyze carnosine, showed no detectable immunoreactive bands. Serum earnosinase appears to be synthesized in the brain, where it is active in the hydrolysis of homocarnosine [1]. This higher primate system may represent a method for regulating the release of GABA in certain neuronal pathways. The three homocarnosinosis siblings have neurological symptoms (dementia and spastic paraplegia); it will be of interest to ascertain the nature of the defect in the molecula. structure of their serum carnosinase. Since most other mammals do not have this enzyme, it will also be interesting to determine the evolutionary antecedent of serum carnosinase. Acknowledgements We thank George R. Lenney and Richard W.N. Child for financial support, Dr. Leslie T a m for discussions, Christopher Locher for help with the ELISA and antigen capture techniques, Sam Perri for rabbit immunization, Alan Komeya for technical assistance, Dr. Ottar Sjaastad for patient serum samples and the Yerkes Regional Primate Research Center for great ape serum samples. References
1 JacksonMC, KuccraCM, LenneyJF. Purificationand propertiesof human serumcarnosinase. Clin Chim Aeta 1991;196:193-205. 2 Sjaastad O, Berstad J, Gjesdahl P, Gjessing L. Homocarnosinosis.2. A familial metabolicdisorder associated with spastic paraplegia, progressive mental deficiency and retinal pigmentation. Acta Neurol Seand 1976;53:275-290. 3 LenneyJF, Peppers SC, Kucera CM, Sjaastad O. Homocarnosinosis:lack ofserum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine. Clin Chim Acta 1983;132:i57-165.
116 4
Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. !11 Quantitation of specific antibodies by enzyme-labeled anti.immunoglobulin in antigen-coated tubes. J lmmunol 1972;109:129-135. 5 Chang SP, Hui GSN, Kato A, Siddiqui WA. Generalized immunological recognition of the major merozoite surface antigen (gp 195) of Plasmodium falciparum. Proe Natl Acad 8ci U.S.A.
1989;86:6343-6347. 6 7 8 9 I0 I1 12 13 14
Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase and activation by cadmium. Clin Chim Acta 1982;123:221-231. Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue carnosinase. Bioehem. J. 1985;228:653-660. Lenney JF. Human cytosolic carnosinase: evidence for identity with prolinas¢, a non-specific dipeptidase. Biol Chem Hoppe-Seyler 1990;371:167-171. Onchterlony O. Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol Microbiol Seand 1953;32:231-240. Schneider C, Newman RA, Sutherland DR, Asset U, Greaves MF. A one-step purification ofmembran¢ proteins using a high efficiency immunomatrix. J Biol Chem 1982;257:10766-10"/69. Higgins RC, Dahmus ME. Rapid visualization of protein bands on 8DS-gels. Anal Biochem 1979;93:257-260. Hunkapiller MW, Lujan E. Purification of microgram quantities of proteins by polyaerylamide gel electrophoresis. In: Shively JE, ed. Methods of protein microcharacterization. Clifton, N J: Humana Press, 1986;89-101. Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988;9:255-262. Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninicacid. Anal Biochem 1985;150:76-85.
109
CCA 05213
Homocarnosinosis patients and great apes have a serum protein that cross-reacts with human serum carnosinase M e l C. J a c k s o n a n d J a m e s F. L e n n e y Pharmacology Department, School of Medicine, University of Hawaii, Honohdu, HI 96822 (USA) (Received 12 August 1991; revision received 30 October 1991; accepted 8 November 1991)
Key words: Serum carnosinase; Dipeptidase; Homocarnosinosis; Great apes; Antigen capture
Summary A specific polyclonal antiserum to human serum carnosinase was raised in rabbits and was used to prepare an agarose-protein A-antibody matrix. An antigen capture procedure showed that sera from homocarnosinosis patients, which lack carnosinase activity, contain an immunoreactive protein (M r 75 000) indistinguishable from the carnosinase band from normal serum. Other higher primates have active serum carnosinase and a similar immunoreactive Mr 75 000 protein. The immunoaffinity matrix was used in a facile procedure to isolate pure carnosinase from human plasma with a yield of 69%. The antiserum inhibited human serum earnosinase strongly, but the maximum inhibition attained averaged only 71%. The antiserum inhibited human and chimpanzee serum carnosinases more effectively than gorilla or other higher primate serum carnosinases.
Introduction Human serum carnosinase is a dipeptidase which is especially active in hydrolyzing carnosine (/~-alanylhistidine) and anserine (/3-alanyl-l-methylhistidine); it also splits homocarnosine (7-aminobutyric acid (GABA)-histidine) [1]. The active enzyme is a dimer, the two subunits (Mr 75 000) being connected by one or more Correspondence to: Mel C. Jackson, Pharmacology Department, School of Medicine, University of Hawaii, 1960 East West Road, Honolulu, H! 96822, U.S.A. Abbreviation: BBS, 0.1 mol/i sodium borate buffer (pH 8.2) containing 0.9% NaCI.
II0
disulfide bonds [1]. Serum carnosinase appears to be secreted by brain cells into cerebrospinal fluid, which transports it into the blood stream [I]. Carnosine hydrolyzing activity is present in higher primate sera, but when 12 non-primate mammals were tested, none exhibited serum activity except the Golden hamster [1]. Homocarnosinosis is a rare human metabolic disorder; Sjaastad et al. found that patients with this disease have cerebral spinal fluid (CSF) homocarnosine concentrations that are elevated 20-fold above normal [2]. These patients lack serum carnosinase activity [3] and this appears to explain the abnormal concentrations of CSF homocarnosine. We now report that the homocarnosinosis patients have an immunoreactive serum protein which appears to be an inactive form of carnosinase. Other higher primates and the Golden hamster also have a serum protein which cross-reacts with human serum carnosinase.
Experimental Materials Protein A-Sepharose 4B and all other chemicals were from Sigma Chemical Co.
Development of polyclonal antiserum Homogeneous human serum carnosinase was prepared from plasma as previously described [1]. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the enzyme band was excised and macerated with an equal volume of Freund's complete adjuvant. Approximately 35 ttg of enzyme was injected into a New Zealand white rabbit intradermally at multiple sites. Two weeks later the rabbit was reinjected with 35 #g ofantigen in the presence of Freund's incomplete adjuvant; this procedure was repeated after 4 weeks. Seven weeks after the initial injections the rabbit was exsanguinated via the femoral artery and the serum was stored in small aliquots at -70°C. Antibody titer was monitored during the immunization period by preincubating 50/zl of test-bleed serum with 150 #1 of a human serum carnosinase preparation for 30 min at 30°C. Aliquots (25 and 50/~i) were removed from the mixture and from control solutions containing enzyme without antiserum; these were assayed for activity against carnosine. Antibody titer was also measured using the enzyme-linked immunosorbent assay of Engvall and Perlmann [4] as modified by Chang et ai. [5].
Enzyme assays Serum carnosinase activity against carnosine and homocarnosine was measured using previously described procedures [6]. Free histidine liberated from the substrate was quantitated fluorometrically after reaction with o-phthaldialdehyde under conditions specific for histidine. Human tissue carnosinase activity against carnosine was measured under conditions optimal for this dipeptidase [7]. It is recommended that this enzyme, which is identical to prolinase, be called non-specific cytosolic
ill
dipeptidase [8]. The inhibitory activity of antiserum was measured after preincubation of enzyme with antiserum at 30°C as described above.
Double immunodiffusion Antibody monospecificity was tested using the double immunodiffusion procedure of Ouehterlony [9], in 1.5% agarose gels.
Preparation of immunoaffinity complex lmmunoaffinity chromatography matrix was prepared essentially as described by Schneider et al. [I0], incubating the antiserum with protein A-Sepharose 4B overnight at 4°C.
Immunoaffinity purification of human serum carnosinase Human plasma was chromatographed on a DEAE-cellulose column [11 and the fractions containing carnosinase were pooled and concentrated by ultrafiltration. Concentrate (15 ml) was mixed with 1 ml of Sepharose-protein A-antibody beads and 30 ml of BBS; the mixture was rotated for 24 h at 4°C. Elution of bound protein [I0] was confirmed by SDS-PAGE (4 mA, 16 h) followed by immersion of the gel in 4 mol/l sodium acetate [I 1]. The major protein band was excised, the gel slice was chopped into l-ram 3 cubes and the enzyme was electroeluted following the method of Hunkapiller and Lujan [12]; eluted enzyme was stored at -70°C.
Antigen capture and visualization Serum samples were assayed for immunoreactivity with serum carnosinase antibody by incubating 100 #1 of serum with 50 tzl of the Sepharose-protein A-antibody beads and 6 ml of BBS while rotating for 16 h at 4°C. After washing in BBS, 50 #i of a 1:1 ethylene glycol-BBS mixture were added to release bound antigen. Eluted antigen was examined by SDS-gel electrophoresis (200 V, 4°C) under reducing conditions in 8.75% polyacrylamide gels; protein bands were visualized using the Coomassie Blue G-250 staining technique of Neuhoffet al. [13]. Molecular weight was estimated using carbonic anhydrase, ovalburnin, bovine serum albumin, phosphorylase b, B-galactosidase and myosin as marker proteins.
Protein assay Protein concentrations were measured using the micro bicinchoninic acid method of Smith et al. [14], with bovine serum albumin as the reference protein. Results and Discussion
Antibody specificity and titer Double immunodiffusion assay &the antiserum showed a single precipitation line
112
with all antigen preparations tested. No spurs were seen at the junction of precipitation lines formed with crude or partially purified antigen preparations, indicating antibody monospeeificity. Enzyme-linked immunosorbent assay (ELISA) of the antiserum against purified serum carnosinase gave a titer of between 1:6250 and 1:31 250. Inhibition of serum carnosinase by antiserum
Figure 1 shows that human serum carnosinase was strongly inhibited by very low concentrations of antiserum. Incomplete inhibition at higher concentrations may be attributable to partial'blocking of the active center by antibodies or to a conformationai change affectin8 the active center. A similar maximum degree of inhibition
25
~
2O
0< .x-:. u.! ~ < zm ~03 O~ Z o
C~
~
lo
5
0
0
t 1
', 2
t 3
I 4
I 5
I 6
I 7
pl ANTISERUM / 0.5 ml DIGEST Fig. I. Effect of increasing amounts of antiserum on human serum carnosinase activity. Aliquots of a partially purified enzyme preparation (40 pl) were diluted to 200 #1 with 125 mmol/I NH4OH/HCI buffer (pH 8.5) containing 0-50 #l of antiserum. After 30 rain at 30*C, two aliquots from each tube were assayed for activity using carnosine as the substrate.
113 TABLE I Inhibition of higher primate serum carnosinases by antiserum to human serum carnosinase Serum source
Human Gorilla Chimpanzee Gibbon Pygmy Chimp
% Inhibition 30 rain
120 rain
42 6 45 0 0
56 22 49 4 0
Mixtures containing 50 pl serum, 100 ~tl 25 mmol/lN-ethylmorpholine/HCibuffer (pH 7.6) and 50 #1 antiserum were preincubated for 30 rain or 120 rain at 30°C. Control mixturescontaining no antiserum were also preincubated at 300C. Two 50-#1 aliquots from each tube were then assayedfor activityagainst carnosine.
was obtained when homocarnosine was employed as the substrate, suggesting that both dipeptides were hydrolyzed in the same active center. Table I shows the effect of the antiserum on the activity of serum carnosinases from five higher primates. The human and chimpanzee enzymes were inhibited to approximately the same extent, the gorilla enzyme was inhibited moderately, whereas little or no inhibition was seen with the gibbon and pygmy chimp enzymes. (Preimmune rabbit serum contained no carnosinase activity and had no inhibitory effect on human serum carnosinase). Evidently the structure o f the chimpanzee serum carnosinase resembles that of the human enzyme more closely than does the structure of the gorilla enzyme. Crude preparations of human kidney cytosolic non-specific dipeptidase [8] were inhibited by the antiserum in their hydrolysis of carnosine by an average of 9.5 ± 3.8% (n --- 4). This may be attributable to contamination of the preparations with serum carnosinase present in trapped blood. Golden hamster is the only non-primate animal known to have a serum carnosinase [1]. Surprisingly, the antiserum inhibited this enzyme 31 ± 4% (n = 4).
Immunoaffinity purification of serum carnosinase Immunoaffinity purification of human serum carnosinase, followed by preparative SDS-PAGE and electroelution from the gel, resulted in an apparently homogeneouS, protein. When this protein was analyzed by SDS-PAGE under reducing conditions, only one band was detected (Fig. 2). The M r of this protein was found to be 75 000 in agreement with the previously determined subunit Mr of human serum carnosinase purified by conventional methods [1]. By this procedure, approximately 550 #g of pure enzyme were obtained from 200 ml plasma, a 69% yield, assuming that plasma contains 4 mg enzyme per liter [ 1 ] .
114
~
..
d
@
.~_
:~
:!:,ii(
~
.~ -
=
f". .z.
._
U
U
~
~
,~
115
Antigen capture The antigen capture procedure was applied to serum samples from the three homocarnosinosis siblings, their mother and father and a normal control. Bound protein was eluted from the immunoaffinity matrix and subjected to SDS-PAGE. As shown in Fig. 3, all six eluates showed a single protein band at M r 75 000, indicating that the homocarnosinosis patients have an immunoreactive but enzymatically inactive form of serum earnosinase. Although the synthesis of this enzyme was defective, it was nonetheless secreted into the blood stream. Double immunodiffusion assay of these samples showed no spur formation when adjacent to normal human serum samples. Thus, the homocarnosinosis enzyme was antigenically identical to the normal enzyme with respect to this antiserum. When the antigen capture procedure was used with four higher primate serum samples, the results shown in Fig. 4 were obtained. A Mr 75 000" band was seen in all lanes, although the band was weak in the case of the pygmy chimp. Evidently these great ape serum carnosinases have the same Mr as the human enzyme and they also cross-react with it. Again, the Ouchterlony assay indicated that the great ape enzymes were antigenicaily the same as the normal human serum carnosinase. Serum from the orangutan showed a faint band at M r 70 000 and Golden hamster serum gave a faint band at Mr 65 000 (not shown). Rat serum, which does not hydrolyze carnosine, showed no detectable immunoreactive bands. Serum earnosinase appears to be synthesized in the brain, where it is active in the hydrolysis of homocarnosine [1]. This higher primate system may represent a method for regulating the release of GABA in certain neuronal pathways. The three homocarnosinosis siblings have neurological symptoms (dementia and spastic paraplegia); it will be of interest to ascertain the nature of the defect in the molecula. structure of their serum carnosinase. Since most other mammals do not have this enzyme, it will also be interesting to determine the evolutionary antecedent of serum carnosinase. Acknowledgements We thank George R. Lenney and Richard W.N. Child for financial support, Dr. Leslie T a m for discussions, Christopher Locher for help with the ELISA and antigen capture techniques, Sam Perri for rabbit immunization, Alan Komeya for technical assistance, Dr. Ottar Sjaastad for patient serum samples and the Yerkes Regional Primate Research Center for great ape serum samples. References
1 JacksonMC, KuccraCM, LenneyJF. Purificationand propertiesof human serumcarnosinase. Clin Chim Aeta 1991;196:193-205. 2 Sjaastad O, Berstad J, Gjesdahl P, Gjessing L. Homocarnosinosis.2. A familial metabolicdisorder associated with spastic paraplegia, progressive mental deficiency and retinal pigmentation. Acta Neurol Seand 1976;53:275-290. 3 LenneyJF, Peppers SC, Kucera CM, Sjaastad O. Homocarnosinosis:lack ofserum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine. Clin Chim Acta 1983;132:i57-165.
116 4
Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. !11 Quantitation of specific antibodies by enzyme-labeled anti.immunoglobulin in antigen-coated tubes. J lmmunol 1972;109:129-135. 5 Chang SP, Hui GSN, Kato A, Siddiqui WA. Generalized immunological recognition of the major merozoite surface antigen (gp 195) of Plasmodium falciparum. Proe Natl Acad 8ci U.S.A.
1989;86:6343-6347. 6 7 8 9 I0 I1 12 13 14
Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase and activation by cadmium. Clin Chim Acta 1982;123:221-231. Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue carnosinase. Bioehem. J. 1985;228:653-660. Lenney JF. Human cytosolic carnosinase: evidence for identity with prolinas¢, a non-specific dipeptidase. Biol Chem Hoppe-Seyler 1990;371:167-171. Onchterlony O. Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol Microbiol Seand 1953;32:231-240. Schneider C, Newman RA, Sutherland DR, Asset U, Greaves MF. A one-step purification ofmembran¢ proteins using a high efficiency immunomatrix. J Biol Chem 1982;257:10766-10"/69. Higgins RC, Dahmus ME. Rapid visualization of protein bands on 8DS-gels. Anal Biochem 1979;93:257-260. Hunkapiller MW, Lujan E. Purification of microgram quantities of proteins by polyaerylamide gel electrophoresis. In: Shively JE, ed. Methods of protein microcharacterization. Clifton, N J: Humana Press, 1986;89-101. Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988;9:255-262. Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninicacid. Anal Biochem 1985;150:76-85.
