AJEBAK 53 (Pt. 1) 77-88 (1975)
PURIFICATION, PROPERTIES AND KINETICS OF SHEEP AND HUMAN RENIN SUBSTRATES by S. L. SKINNER, ]. R. DUNN, I. MAZZETTI, D. I. CAMPBELL AND N, H. F I U G E " (From the Department of Physiology, University of Melbourne, Parkville, Vietoria, Australia 3052.) (Accepted for publication September 25, 1974.) Summary. Sheep plasTiia renin substralf was purified 1,200-fold l>y usinjj nephrfctomised sheep plasma, followed by DEAE-Sephadex chromatojijraphy and gel filtration. Th? purified substrate contained 8 nji angiotensin Il/nig protein and had an estimated molecular vveijjht of 52,000. The kinetic characteristics of the purified substrate were identical both to those of tuipurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7-5, K,,, of the human renin-sheep substrate reaction was 0-29 [iM and for sheep reninsheep substrate. 2-0 fiM. Sheep substrate was .susceptible to peptic diHeslion with generation of pepsitenshi. Hunuin renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0-9 ng angiotensin Il/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82.000) more anionic protein than sheep substrate and was resistant to the proteolytit actions of both pepsin and shi-ep renin. K,,, for the human renin-human substrate reaction was high and conid not be accurately detennined (range 3-8 nM. mean 5-7 ^iM). The presence of human substrate in a human rcnin-sheep substrate system did not alter the measured initial velocity. In both shet'p and man, the nonnal concentration (»f ri^nin substrate is considerably less than K,,, and must therefore be considered a dtterminant of angiotensin production rate j>i vivo. Key words: Renin kinetics, pepsitensin. Renin substrate after nephrectomy.
INTRODUCTION. Following bilateral nephrectomy in experimental animals, the plasma concentration of the specific protein substrate for renin increases several-fold (Page and McCubbin, 1969). Because of its high substrate concentration, absence of 'Present address: Department of Clinical Science, John Curtin School of Medical Research, Australian National University. Canberra, A.CT.. Australia.
78
SKINNER, DUNN, MAZZETTI, CAMPBELL AND F I D G E
endogenous renin and plentiful supply, 6-day nephrectomised sheep plasma has been used to achieve zero-order kinetics for the quantitative assay in vitro of human renin {Skinner, 1967; Stockigt, ColUns and Biglieri, 1971). In those studies it became apparent that whereas the concentration of sheep renin substrate in neat nephrectomy plasma was more than adequate to saturate the human enzyme, much higher concentrations of the homologous human substrate were incapable of achieving this effect (Skinner, Lumbers and Symonds, 1969; 1972). This observation indicated considerable differences in the affinity of human renin for each substrate. In the present study, with purer reactants, correlates have been sought between the kinetic constants of the renin-renin substrate reaction in sheep and man and tho physico-chemical properties of each substrate. The findings have relevance to the physiological function of the reninangiotensin system in each species. In addition, the work has led to the isolation by simple techniques of a highly purified stable renin substrate from sheep plasma which has been radio-labelled and used in a sensitive quantitative assay of human plasma renin (Campbell and Skinner, 1971).
MATERIALS AND METHODS.
Renin substrate. Sheep renin substrate was obtained from both norma! and nephrectomised sheep. Twelve sheep (ewes and wethers) were anaesthetised, bilaterally nephrectomised and allowed to recover. Six days later the animals were lightly anaesthetised with Nembutal and exsanguinated by arterial haemorrhage after intravenous injection of 10.000 units of heparin. Furths^r heparin was added to the collected hlnod to provide a final estimated concentration of 10 units/ml blood. Up lo 3 I of blood were obtained from each animal. The plasma was separated by ccntrifugation at 4° and stored frozen at —15°. During the 6-d.iy period the sheep remained active, displaying little evidence of their condition other than a decrease in food intake and some scouring. Plasma samples were collected daily dnring the 6-day period from 5 of the sheep. Human renin .sulwtrate was prepared from plasma collected by vcnipuncture from normal male and female volunteers and from healthy pregnant women at 36-40 weeks' gestation. The latter source was exploited for its high concentration of renin substrate (Skinner et al. 1972). Heparin (10 units/ml) was added to all blood, the plasma removed, pooled into separate noimal or pregnancy stocks and stored at —IS". Purification of renin s
DEAE-Sephadex chroinutonrapluj. 50-100 nil of whole plasma were dialysed for 18 h at 4° against 5 I of 0 02M-sodium phosphate buifcr pH 7-5 and then applied to a column (100 cm X 2-5 cm) of DEAE-Sephadex A50 eciuilibrated against the same buffer. A linear gradient was developed by introducing 500 ml of 0-5M sodium chloride—0 02M-sodiuni phosphate bufler pH 7-5 into a mixing ehamber containing 500 ml of the equilibration buffer. Eluate fraetions (10 uil) containing the substrate were then combined antl concentrated by ultrafiltration through either Visking casings or Diaflo membranes, prior to storage at —15°. Gel filtTation. 10 ml of the concentrated eluate from DEAE-Sephadex column chromatography were dialysed for 18 h at 4° agahist 5 1 of 0-02M-sodium phospbate buffer pH 7-5 and then applied to a column (200 cm x 2 5 cm) of Sephadex G-lOO or G-200 equilibrated and eluted into 5 or 10 ml fractions with the same buffer at 4°. at a constant flow rate (20-30 ml/h). The cluate was scanned for absorption at 280 nm. Growth of micro-organisms was suppressed by addition of 0-002!! chlorhexidine gluconate to the bufler solutions used to eciuilibrate the Sephadex eolumns.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
79
Determination of inotccutar weight. Molecular weight of renin substrate was detemiined by gel filtration by the method of Andrews (1964). Sephadex columns (100 cm x 2 4 cm) were equilibrated and eluted with 0-{)2M sodium phosphate buffer pH 7-5 at 4°. The following crystalline proteins served as molecular weight markers: lysozyme (mol. wt. 14.300); cytochrome c (mol, wt. 12,700); bovine serum albumin (mol. wt. 66,000); egg albumin (mol. wt. 43,000); human gammaglobulin (mol. wt. 156.000). Sephadex G-lOO was u.sed for sheep substrate and Sephadex G-200 was used for human substrate. Protein concentration was measured by the l)iurft method (Layne, 1957). Hfuin. Sheep renal renin was kindly pn)\ided by the Howard Florcy Institute of Experimental Physiology and Medicine, Parkville, Australia.' Human renin ciiiuc from two sources. For kinetic studies the WHO standard renal renin was employed as snpplied by the Division of Biological Standards, National Institute for Medical Research, Mill Hill. Amounts are expressed in Goldblatt Units. For estimation of botb human and sheep substrates, human renal renin prepared by trichloroacetic acid precipitation (Brown et al., 1964) was used. All reniii preparations were shown to be angiotensinase-free by better than 90% survival of added angiotensin II-/J-amide (Ciba) over 24 h at pH 7-5 at 37°. Angiotansin assay. Angiotensin was measured by bioassay on rat blood pressnre by the l>racket method (Brown et at., 1964) assaying against synthetic angiotensin II-/a-aniide (Ciba) witb periodic checks on the relative sensitivity of the preparation to angiotensin I. Prior to injection into the rat the sample for a.ssay was heated at 70° for 5 miii to denature the renin. Renin .substrate assaij. Both sheep and human substrates were estimated as angiotensin released in angiotensinasefree systems during a 30 min incubation with 0-5 Goldblatt Units/ml human renin at pH 7-5, at 37°. No furtiier angiotensin was generated during more prolonged incubations. Plasma or column eluates were diluted prior to substrate estimation such that generated angiotensin remained below 200 ng/ml in the incubation mixture. Plasma was rendered angiotensinase-free by dialysis for 18 h at 4" against 5 1 pH 4-5 buffer (0-02SM-citric acid, 0 045M-di-sodium bydrogen pbosphate, 0 082M-sodium chloride) containing 5niM EDTA, incubation at 32° for 30 min followed by dialysis for 18 h at 4° against .5 1 pH 7 5 buffer (0-lM-sodium phosphate, 0-075M-.sodium chloride) containing 1 niM EUTA (Skinner, 1967). Molar concentration of substrate was obtained by assuming that one mole of angiotensin 11 (mol. wt. 1.045) is released from one mole of the protein substrate. Thus, 1 ^g substrate exiiressed as angiotensin II derives from 1 nmole snbstratc. At a substrate concentration of 1 ng angiotensin/ml the concentration could also be expressed as 1 M,M. Kinetic studies. The influence of substrate concentration on reaction velocity was studied for both human and sheep renin substrates reacting with both human and sheep renins at 37° at pH 7-5. Veloeity/substrate plots were constructed from serial dilutions of substrate incubated with ctmstant amounts of renin. K,,, and V^^^^ were determined by regression analysis of the unweighted data using Lineweaver-Burk or Ilofstee plots (Uixon and Webb, 1964). Since no more than 10% of the substrate was consumed in these incubations, tbe measured velocity was considered not to differ significantly from initial velocity (Dixon and Webb, 1964). Maxinuun rates of angiotensin formation were kept at or below 100 ng/ml/h. Higher rates could not be accurately determined. r II Piepared bv Dr. Ralph Peterson in a large-scale method employing alcohol precipitation followed by gel filtration (unpublished).
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SKINNER, DUNN, MAZZETTI, CAMPBELL AND F I D G E
Studies were performed using both neat plasma and purified substrate. Plasma was rendered angiotensinase-free as described for substrate as.say. Prior to kinetic studies, purified substrate preparations were dialysed for 18 h at 4° against 5 I of 0-lM-sodium phosphate— 0-075M-sodiuin chloride buffer pll 7-5 containing 1 mM EDTA. This same buffer was used for dilution of substrate and renin. Gomplete survival of reactants and the end product was confirmed during all incubations. Serial dilntions of substrate without added renin were also studied as controls for the presence of endogenous renin.
RESULTS. Renin substrate in neat plasma. Expressed us angiotensin II content, the ineiin substrate level in normal sheep was 0-41 ± 0 03 fig/ml {± SE, n ^ 5) rising to 2-45 ± 0-25 /xg/ml (± SE, n = 12) at day 6 post-nephrectoniy. Specifie aetivity with respect to total protein rose 5- to lO-fold (maxinuun 0 0 4 /xg/mg protein) in different sheep. In the 5 sheep in which it was studied there was a progressive increase in plasma substrate concentration over the 6 days following nephrectomy without evidence of a plateau being reached. Renin stibstrate concentration in pooled normal male and female human plasma was 1 jLig/m! and in pooled pregnancy plasma was 7 /ig/ml. The specific activity of the pregnancy plasma was 0-09 /ig angiotensin/mg protein. DEAE-Sephaclex chromatograpinj. From five separate columns, renin substrate in nephrectomi.sed sheep plasma eluted as a single, smooth peak between 0-lOM- and 0-15M-sodium chloride at the beginning of the albumin peak. Renin substrate from normal sheep plasma eluted in the same position. Recovery was 90-100^ and specific activity increased up to lO-fold (reaching 0-33 jUg angiotensin/mg protein) in fractions from the first half of the peak. This chromatographed substrate was free of angiote-nsinase. The elution position of angiotensinase was not determined. Human substrate also eluted as a single peak, but in contrast to sheep substrate its elution was between 0-22M- and 0 25M-sodium chloride at the end of the albumin peak. Substrate in plasma from both pregnant and non-pregnant subjects behaved identical!). Recovery was 40-50% and the increase in specific activity varied from 2- to 12-fold in different experiments. DEAE-Sephadex cohmm ehromatography was effective in producing a human substrate preparation free of angiotensinase with low endogenous renin activity. Renin eluted between 0-18M- and 0 22M-sodium chloride, in front of the albumin and the substrate peaks. The highest specific activity human substrate obtained with this system was 0-9 yxg angiotensin/mg protein. The relative elution positions of sheep and human substrates from a mixture of each is shown in Fig. 1. In such an experiment, each substrate could be identified by incubating the fractions with sheep renin which released angiotensin from only the homologous stibstrate, whereas human renin acted upon both substrates.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
81
Both chromatographed substrate.s were stable during incubation at 37° and were resistant to freezing and thawing.
FRACTION NUMBER
Fig. 1. Ehition pattern of sheep and hnnian renin substrates froiu a mixtnre of sheep and human plasma, on a DEAE-Sephadex A50 column (100 cm x 2-5 cm) e(iuilii)rate(l with 0-02M-sodium nhospluite bufTer pH 7 5 and ehited with a linear gradient of sodium chloride. Fractions of volmnt- 10-0 ml were collected. Continnous trace is the nncaiibrated al).sorbance scan at 280 nm. Sheep sulxstrate ( A ) and human substrate ( • ) elute at 0-12 aud 0'24M-sodium chloride respectively.
Gel filtration chromatography of post-DEAE-Sephadex fractions. On Sephadex C-200 httle further increase in specifie aetivity of either sheep or human snbstrate was achieved above that obtained on DEAE-Sephadex. On Sephadex G-lOO (Fig. 2), sheep but not human substrate was widcl\' separated (80 ml) from the protein peak with high recovery (90-100%). Specific activity of sheep substrate in four separate experiments reached 8, 6, 5 and 5 /ig angiotensin/mg protein. This substrate preparation was stable when stored at -15° and was not affected by thawing, but lost activity over 4 days at 4°. Human substrate did not penetrate the gel phase of C-lOO Sephadex and no further increase in specific activity was achieved. Molecular weight. On Sephadex C-100, sheep substrate behaved as a molecule smaller than ijovine serum albumin monomer with a mol. wt. of 52,000. Sephadex G-200 was required for mol. wt. estimations of human substrate. It behaved as a molecule larger than bovine sernm albumin monomer with a mol. wt. of 82,000. Suhstrate specific activity. Calculating from the estimated molecular weights and assitniing that one mole of angiotensin is released from one mole of protein substrate, the highest specifie aetivity obtained for sheep substrate was 0-4 g substrate/g protein (8 nmole/mg protein) and for huuian substrate was 0-07 g substrate/g protein (0-9 nmole/mg protein).
82
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE
Kinetics. (i) Human renin-sheep suhstrate. Figure 3 shows velocity/substrate plots for both neat plasma from nephrectomised sheep and the purified sheep substrate. The only difference between the two curves is the plateau in velocity at the high protein concentrations of neat plasma. Omitting the plateau, linear transformation of this data yielded identical K,,, values for each substrate. The mean of four separate experiments was 0 29 /xM (0-21, 0 24, 0 29, 0-40).
FRACTION NUMBER
Fig. 2. Gei filtration on a Sephadex G-lOO phosphate buffer pll the uncalibrated
of sheep substrate after prior DEAE-Sephadex column chroinatiigraphy, column (100 cm x 2'5 em) equilibrated and eluted with 0-02M-sodimn 7-5. Fractions i)f volume 10-0 Till were collected. C:ontiiiuons trace is abs()ri)auce at 280 lun. Sheep substrate ( • ) elnted after 210 nil.
RENIN SUBSTRATE CONCENTRATION
Fig. 3. Eftect of sheep renin substrate concentration ou initial velocity of reaction with human renin (8 x 10-^ Goldblatt Units/ml). Abscissa: Rate of substrate degradation where 1 nniole/l/h is equivalent to an angiotensiu II generation rate of 1 HE/Vh. Specific activity of purified suhstrate ( • ) was S rmiole/mg protein and for neat nephrectomy plasma ( O ) 0-05 iimole/ing protein.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
83
(ii) Human remn-human .nibnirate. Human sulistrate from normal plasina, pregnancy plasma and from DEAE-Sephadex column chromatography all showed similar reaction kinetics (Fig. 4). The highest specific activity hnman substrate used in this study contained onl\- 0 14 /xg angiotensin/mg protein (0-14 nmole/mg) at the highest concentration of 5-6 /Ag angiotensin/ml. At this concentration, Vmax was not approached and the K,,, derived from linear transformation of the data is accordingly subject to considerable error. In separate experiments K,,, varied widely at 7 8 , 7-5, 5-0 and 2-8 /IAM (mean 5 7 fxWl). The difficulty of consistently preparing high specific activity human substrate free of endogenous renin prevented more accurate determinations.
RENIN SUBSTRATE CONCENTRATION (/.M)
Fig. 4. Effect of human and sheep renin substrate concentration nn initial reaction veloeity with human renin ( 8 x 10--I Goldljlatt Units/inl). Abscissa: Rate of substrate degradation where I nmole/f/h is equivalent to an angiotensin gc'neration rate t)f 1 n g / l / h . Lower curve; human substrate preparations. Post-DKAE-Sepliadex human substrate ( O ) - Neat pregnancy plasma ( • ) . Plasma from women takinj; oral contraceptives ( • ) . Pooled normal human plasma ( A ) . Upper cnr\e: purified sheep substrate ( • ) . Means ± SE, n = 4.
(iii) Hutnan renin in a mixture of human and sheep substrates. Hnman pregnancy plasma at the constant renin substrate concentrations of either 1-0 or 2 7 ^g angiotensin/ml was mixed with serial dilutions of sheep substrate from 3-0 down to 0 25 /ig angiotensin/ml. Initial velocity was measured both with and without the addition of 10" •' Goldblatt Units of human renin/ml of incubate. Initial velocity was also measmed in control incubates containing the same concentrations of sheep substrate and renin bnt without added human plasma. At all sheep substrate concentrations in the mixed substrate system, total reaction rate was not altered by the presence of human substrate.
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE IV Sheep renin-sheep suhstrate. Sheep sul)strate, whether as unpurified nephrectomised phisma or after purification, displayed similar reaction kinetics (Fig. 5). K,,, for both reactions was 2-0 fiM. Unpurified normal sheep plasma substrate possessed identical kinetic characteristics to those of nephrectomised sheep plasma substrate and the purified substrate. For this comparison the higher specific activity substrates were diluted with buffer until substrate concentration equalled that of normal plasma. Initial velocity with added renin was then identical in all three substrate preparations.
0
2
4
6
RENIN SUBSTRATE CONCENTRATION (^Wj
Fig. 5. Lower curve shows effect of sheep renin substrate concentration on initial reaction veloeity with sheep renin. Purified substiate (•). Neat nephreetomy plasma (A). Nonnal sheep plasma (O)- ^a, = 2 \i\\. \',,,ax = 100 nmole/I/h. Upper curve is a derived plot of ths human renin-sheep suhstrate reaction assuming a Vj^j^jj of 100 nmole/I/h and K^n of 0-29 jiM.
Formation of angiotensin from renin substrates by pepsin. Pepsin is known to act on ox and horse substrates to form angiotensin II (de Fernandez, Paladini and Deluis, 1965). Figure 6 shows that whereas pepsin (Sigma, hog pepsin, twice cr\'stallized) acts on sheep substrate at pH levels 4-0 and 6-5, no angiotensin forms from human substrate under the same conditions, Human renin continued to release the same amount of angiotensin from human substrate after prior incubations of the substrate with pepsin (Fig. 5), indicating that the human substrate was resistant to proteolysis by this enzyme. With nephrectomised sheep plasma, pepsin (2 mg/ml) formed angiotensin at all pH levels between 2 and 6-5. Above pH 7-0 no formation occurred. Below pH 4 0 loss of formed angiotensin was rapid. At pH 6-6-5 no loss of
SPECIES DIFFERENCES IN RENIN SUBSTRATE
85
either formed, or added angiotensin, occurred during 60 min incubations with pepsin, and the amount generated was not significantly different from the maximum vield with renin itself.
\ HUMAN
P E P S I N PH 5 . 0
7 HUMAN
R E N I N P H 7 . 5
R E N I N
PH 7 . 5 Fig. 6. Actioii of lioy pepsin on sheep and human renin substrates. Rat blood pressurt' assay against standard angiotensin II e.xpressed in nanograms. Nephrectomised sheep plasma (Sheep). Pregnancy plasma (Ihnnan). Concentration of pepsin, 2 nig/ml; Human renin, 0-5 Coldblatt Units/ml. Incubation time 60 min.
Human substrate in Colin fractions. Fractious IV-4, IV-5 and IV-6 (Commonwealth Serum Laboratories, Melbourne) contaiTied respectively 0 008, 0 007 and 0 /ig angiotensin/mg protein. Such low specific activities made these fractions of less use than pregnancv plasma for purification purposes. DISCUSSION. Chromatography on DEAE-Sephadex followed by Sephadex G-lOO yielded a renin substrate from nephrectomised sheep plasma with specific activity 1,200 times (8 nmole/mg protein) that of normal sheep pUusma. A specific activity equivalent to this was achieved with hog renin substrate by Skeggs et al (1963) but the procedvn-es and losses were far more extensive. At all stages of purification there was no evidence for heterogeneity of sheep substrate as reported for hog substrate (Skeggs ct al, 1963) and supported iu preliminary studies by Dahlheim et al (1969). Reaction kinetics of sheep renin reacting with purified or unpurified sheep substrates, whether from normal or nephrectomised animals were not appreciablv different. This suggests identity of substrates. The K,,, for the homologous reaction in sheep (2 0/iM) was higher than for the heterologons humau renin.sheep substrate reactiou (0-29 ^M) indicating a much higher affinity between the latter reaetants. This property has been exploited in the development of a human plasma renin assay using sheep substrate where zero-order kinetics are achieved (Skinner, 1967; Stockigt et al. 1971). The K,,, for the heterologous reaction lies within the range 0 1 5 to 1 3 ^M found for a variet\' of animal
86
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE
substrates reacting with hnnian renin (Brown et al, 1964; Could, Skeggs and Kahn, 1966; Dahlheim, Weber and Walter, 1970; Wuldhausl and Lewandowski, 1973). Human substrate behaved very differently from sheep substrate in the same chromatographic systems. It appeared to be a more anionic and larger protein than sheep substrate. The molecular weight of 82,000 is cousiderably greater thau that of sheep (52,000) or hog (57,000) substrates (Skeggs et al, 1963). However, the validit) of the present method might be (juestioued, since, as has been shown for some glycoproteins, elution from Sephadex is not always predictable from molecular weight (Andrews, 1965). Skeggs et al (1963) have shown that hog substrate has a small carbohydrate moiety which, if considerably larger in human substrate, might account for the displacement on Sephadex. Differences from sheep substrate were also found in the resistance of hnman substrate to the actions of both pepsin and sheep renin and also the high K,,. for the humau renin-human substrate reaction. Again, such differences might be due to a large carbohydrate moiety. Difficulty was experienced in consistently preparing a human substrate of high specific activity, but the single process of DEAE-Sephadex chromatography yielded some fractions with higher purity (0-9 nmole/mg protein) than achieved in previous work (0 5 nmole/mg protein; Rosenthal et al, 1971). Difficulty in purifviug humau substiate prevented accurate kinetic studies being performed bv conventional methods since V,,,,u was not approached. The mean K^ value of 5 7 jLiM contrasts with such values as 018.5 /iM (Haas and Coldblatt, 1967), 0-07/iM (Poulsen, 1968), 0155/iM (Roseuthal et al, 1971), 0-931 fxW (Could and Creen, 1971), 1-1 /xM (Krakoff, 1973) and 0-4 fxM (Favre and Vallotton, 1973) obtained in studies iu whicli concentration and specific activity of substrate were lower than in the present work. High values are, however, apparent in the publications of Helmer and Judson (1967), Skinner et al (1969, 1972) and Waldhansl and Lewandowski (1973). Skeggs et al (1968) described K,,, values between 3 6 and 50 /xM for a series of short linear synthetic peptide substrates reacting with humau renin but their findings do not assist in understanding the basis for the low affinity between the human reactants, because, for native substrates, affinity is likely to depend to some extent upon protein structure at a distance from the hydrolytic site. Studied at pH 7 4, K,,, values for the homologous reaction in rats (2-4 /iM; Oith et «/„ 1971) and rabbits (2 3 /xM; Campbell et al, 1973) are similar to the present finding for sheep (2 0 fiM). All of these values are considerably less than for the human and raise the possibility that a low affinity renin substrate might offer some evolutionary advantage for man. The finding that in a two substrate system the presence of human substrate did uot reduce reactiou rate between hnman renin and sheep substrate has also been reported by Stockigt et al (1971). In the presence of marked differences in initial velocities with each substrate, failure of hnman suhstrate to reduce reaction velocity in a two substrate system confirms that affinity between the homologous reactants is relatively low. The further possibility, tliat V,,,,,, with
SPECIES DIFFERENCES IN RENIN SUBSTRATE
87
both sheep and humau substrates is the same, could only be tested with purer preparations of human substrate. If this proves to be so, K,,, for the human reaetants could be accurately calculated from the data in Fig. 4, using the Michaelis equation. A value of 7 3 (xW is then predicted. To date the wide range of K,,, values reported for the human reaotants has precluded general agreement on the influence of substrate concentration on angiotensin production in vivo. Differences in the conditions of incubation or assay methods arc not obviously the cause of this and other factors are possible. The problem is exemplified by the recent report of Favre and Vallotton (1973) that affinity of humau renin for renin substrate varies inversely with reaction velocity. Such a fiuding defies any simple explanation including that offered by the authors of the presence of a competitive inhibitor in the plasma snbstrate preparation. No snch anomalies were seen in the present stndy and we conclude that in sheep and man the normal concentration of plasma substrate is at or near the first order range. Substrate concentration in vivo is therefore as important as enzyme concentration in determining production rate of angiotensin. In man it has been established that substrate levels increase in severe hvpertension (Conld and Creen, 1971), duriug pregnancy (Helmer and judson, 1967) and with oral contraceptives (Skinner et al, 1969). Indeed, renin substrate coucentration in al! species thus far studied (BUujuier, 1965; Ryan and McKenzie, 1968) is in a position to exercise control over angiotensin productiou and must therefore be considered an important determinant of circulator)- homeostasis. Acknowledgements. We (.-xprcs.s our thaiiks to Dr. Margaret Smith of the University Department of Obstetrics and Gynaecology. Royal Women's Hospital, Melljourne, for assistance with the collection of human plasma samples, and also to Dr. D. Denton and to the late Dr. J. Goding for providing facilitie.s for the collection of sheep blood. This work was supported l)y a grant from the National Health and Medical Research Council of Australia, REFERENCES. ANDHEWS, P. (1964): 'Estimation of the molecular weights of proteins by Sephadex gel-filtration.' Biochem. /., 9t, 222. ANDREWS. P. (1965): 'The gel-filtration of proteins related to their molecular weights over a wide range.' Biochem. /., 96, 595. , P. (1965): 'Kinetic studies on renin-angiotensinogen reaction after nephrectomy.' Am. } . PhysioL. 208, 1083. BROWN, J. J.. DAVIES, D . L., LEVER. A. F., ROBERTSON, J. I. S., and TREE, M .
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'Influence of high renin substrate levels on renin-anfiiotensin system in prejinancy.' AIM. /. Ohstet. Gynec. 99. 9. KRAKHFF, L, R. (1973): 'MsasureniL'iit of plasma renin substrate by radioimmunoassay of angiotensin I: eoncentrations in syndromes associated with steroid excess.' /. ctin. Eiidoer. Metah., 37, 110. LAYNE, E . G. (1957): "Methods in Enzymology". S. P. Colowick and N. O. Kaplan (editors). Academic, New York, 3, 450. ORTH. H., KHAHE. P.. STEIC;ELMANN, C ,
and GROSS, F . (1971): 'An impro\ed method for the determination of renin in rat kidneys.' P/(iif,'er,s Arc7i.. 329, 125. PAGE, 1. H., and MCGUHBIN, J. W. (eds.)
(1969): "Renal Hypertension". Year Book, Chicago, p. 20. PouLSEN, K. (1968): 'Measurement of renin in human plasma.' Scand. } . elin. Lab. Invest.. 21, 49.
RYAN, J. W.. and MCKENZIE, J. K. (1968):
'Properties of renin substrate in rabbit plasma with a note on its assay.' Biochem. }.. 108. 687. SKEGGS. L. T., LENTZ, E . K., IIOCTHSTRASSEH,
H., and KAHN, J. R. (196.3): 'The purificati
PURIFICATION, PROPERTIES AND KINETICS OF SHEEP AND HUMAN RENIN SUBSTRATES by S. L. SKINNER, ]. R. DUNN, I. MAZZETTI, D. I. CAMPBELL AND N, H. F I U G E " (From the Department of Physiology, University of Melbourne, Parkville, Vietoria, Australia 3052.) (Accepted for publication September 25, 1974.) Summary. Sheep plasTiia renin substralf was purified 1,200-fold l>y usinjj nephrfctomised sheep plasma, followed by DEAE-Sephadex chromatojijraphy and gel filtration. Th? purified substrate contained 8 nji angiotensin Il/nig protein and had an estimated molecular vveijjht of 52,000. The kinetic characteristics of the purified substrate were identical both to those of tuipurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7-5, K,,, of the human renin-sheep substrate reaction was 0-29 [iM and for sheep reninsheep substrate. 2-0 fiM. Sheep substrate was .susceptible to peptic diHeslion with generation of pepsitenshi. Hunuin renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0-9 ng angiotensin Il/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82.000) more anionic protein than sheep substrate and was resistant to the proteolytit actions of both pepsin and shi-ep renin. K,,, for the human renin-human substrate reaction was high and conid not be accurately detennined (range 3-8 nM. mean 5-7 ^iM). The presence of human substrate in a human rcnin-sheep substrate system did not alter the measured initial velocity. In both shet'p and man, the nonnal concentration (»f ri^nin substrate is considerably less than K,,, and must therefore be considered a dtterminant of angiotensin production rate j>i vivo. Key words: Renin kinetics, pepsitensin. Renin substrate after nephrectomy.
INTRODUCTION. Following bilateral nephrectomy in experimental animals, the plasma concentration of the specific protein substrate for renin increases several-fold (Page and McCubbin, 1969). Because of its high substrate concentration, absence of 'Present address: Department of Clinical Science, John Curtin School of Medical Research, Australian National University. Canberra, A.CT.. Australia.
78
SKINNER, DUNN, MAZZETTI, CAMPBELL AND F I D G E
endogenous renin and plentiful supply, 6-day nephrectomised sheep plasma has been used to achieve zero-order kinetics for the quantitative assay in vitro of human renin {Skinner, 1967; Stockigt, ColUns and Biglieri, 1971). In those studies it became apparent that whereas the concentration of sheep renin substrate in neat nephrectomy plasma was more than adequate to saturate the human enzyme, much higher concentrations of the homologous human substrate were incapable of achieving this effect (Skinner, Lumbers and Symonds, 1969; 1972). This observation indicated considerable differences in the affinity of human renin for each substrate. In the present study, with purer reactants, correlates have been sought between the kinetic constants of the renin-renin substrate reaction in sheep and man and tho physico-chemical properties of each substrate. The findings have relevance to the physiological function of the reninangiotensin system in each species. In addition, the work has led to the isolation by simple techniques of a highly purified stable renin substrate from sheep plasma which has been radio-labelled and used in a sensitive quantitative assay of human plasma renin (Campbell and Skinner, 1971).
MATERIALS AND METHODS.
Renin substrate. Sheep renin substrate was obtained from both norma! and nephrectomised sheep. Twelve sheep (ewes and wethers) were anaesthetised, bilaterally nephrectomised and allowed to recover. Six days later the animals were lightly anaesthetised with Nembutal and exsanguinated by arterial haemorrhage after intravenous injection of 10.000 units of heparin. Furths^r heparin was added to the collected hlnod to provide a final estimated concentration of 10 units/ml blood. Up lo 3 I of blood were obtained from each animal. The plasma was separated by ccntrifugation at 4° and stored frozen at —15°. During the 6-d.iy period the sheep remained active, displaying little evidence of their condition other than a decrease in food intake and some scouring. Plasma samples were collected daily dnring the 6-day period from 5 of the sheep. Human renin .sulwtrate was prepared from plasma collected by vcnipuncture from normal male and female volunteers and from healthy pregnant women at 36-40 weeks' gestation. The latter source was exploited for its high concentration of renin substrate (Skinner et al. 1972). Heparin (10 units/ml) was added to all blood, the plasma removed, pooled into separate noimal or pregnancy stocks and stored at —IS". Purification of renin s
DEAE-Sephadex chroinutonrapluj. 50-100 nil of whole plasma were dialysed for 18 h at 4° against 5 I of 0 02M-sodium phosphate buifcr pH 7-5 and then applied to a column (100 cm X 2-5 cm) of DEAE-Sephadex A50 eciuilibrated against the same buffer. A linear gradient was developed by introducing 500 ml of 0-5M sodium chloride—0 02M-sodiuni phosphate bufler pH 7-5 into a mixing ehamber containing 500 ml of the equilibration buffer. Eluate fraetions (10 uil) containing the substrate were then combined antl concentrated by ultrafiltration through either Visking casings or Diaflo membranes, prior to storage at —15°. Gel filtTation. 10 ml of the concentrated eluate from DEAE-Sephadex column chromatography were dialysed for 18 h at 4° agahist 5 1 of 0-02M-sodium phospbate buffer pH 7-5 and then applied to a column (200 cm x 2 5 cm) of Sephadex G-lOO or G-200 equilibrated and eluted into 5 or 10 ml fractions with the same buffer at 4°. at a constant flow rate (20-30 ml/h). The cluate was scanned for absorption at 280 nm. Growth of micro-organisms was suppressed by addition of 0-002!! chlorhexidine gluconate to the bufler solutions used to eciuilibrate the Sephadex eolumns.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
79
Determination of inotccutar weight. Molecular weight of renin substrate was detemiined by gel filtration by the method of Andrews (1964). Sephadex columns (100 cm x 2 4 cm) were equilibrated and eluted with 0-{)2M sodium phosphate buffer pH 7-5 at 4°. The following crystalline proteins served as molecular weight markers: lysozyme (mol. wt. 14.300); cytochrome c (mol, wt. 12,700); bovine serum albumin (mol. wt. 66,000); egg albumin (mol. wt. 43,000); human gammaglobulin (mol. wt. 156.000). Sephadex G-lOO was u.sed for sheep substrate and Sephadex G-200 was used for human substrate. Protein concentration was measured by the l)iurft method (Layne, 1957). Hfuin. Sheep renal renin was kindly pn)\ided by the Howard Florcy Institute of Experimental Physiology and Medicine, Parkville, Australia.' Human renin ciiiuc from two sources. For kinetic studies the WHO standard renal renin was employed as snpplied by the Division of Biological Standards, National Institute for Medical Research, Mill Hill. Amounts are expressed in Goldblatt Units. For estimation of botb human and sheep substrates, human renal renin prepared by trichloroacetic acid precipitation (Brown et al., 1964) was used. All reniii preparations were shown to be angiotensinase-free by better than 90% survival of added angiotensin II-/J-amide (Ciba) over 24 h at pH 7-5 at 37°. Angiotansin assay. Angiotensin was measured by bioassay on rat blood pressnre by the l>racket method (Brown et at., 1964) assaying against synthetic angiotensin II-/a-aniide (Ciba) witb periodic checks on the relative sensitivity of the preparation to angiotensin I. Prior to injection into the rat the sample for a.ssay was heated at 70° for 5 miii to denature the renin. Renin .substrate assaij. Both sheep and human substrates were estimated as angiotensin released in angiotensinasefree systems during a 30 min incubation with 0-5 Goldblatt Units/ml human renin at pH 7-5, at 37°. No furtiier angiotensin was generated during more prolonged incubations. Plasma or column eluates were diluted prior to substrate estimation such that generated angiotensin remained below 200 ng/ml in the incubation mixture. Plasma was rendered angiotensinase-free by dialysis for 18 h at 4" against 5 1 pH 4-5 buffer (0-02SM-citric acid, 0 045M-di-sodium bydrogen pbosphate, 0 082M-sodium chloride) containing 5niM EDTA, incubation at 32° for 30 min followed by dialysis for 18 h at 4° against .5 1 pH 7 5 buffer (0-lM-sodium phosphate, 0-075M-.sodium chloride) containing 1 niM EUTA (Skinner, 1967). Molar concentration of substrate was obtained by assuming that one mole of angiotensin 11 (mol. wt. 1.045) is released from one mole of the protein substrate. Thus, 1 ^g substrate exiiressed as angiotensin II derives from 1 nmole snbstratc. At a substrate concentration of 1 ng angiotensin/ml the concentration could also be expressed as 1 M,M. Kinetic studies. The influence of substrate concentration on reaction velocity was studied for both human and sheep renin substrates reacting with both human and sheep renins at 37° at pH 7-5. Veloeity/substrate plots were constructed from serial dilutions of substrate incubated with ctmstant amounts of renin. K,,, and V^^^^ were determined by regression analysis of the unweighted data using Lineweaver-Burk or Ilofstee plots (Uixon and Webb, 1964). Since no more than 10% of the substrate was consumed in these incubations, tbe measured velocity was considered not to differ significantly from initial velocity (Dixon and Webb, 1964). Maxinuun rates of angiotensin formation were kept at or below 100 ng/ml/h. Higher rates could not be accurately determined. r II Piepared bv Dr. Ralph Peterson in a large-scale method employing alcohol precipitation followed by gel filtration (unpublished).
80
SKINNER, DUNN, MAZZETTI, CAMPBELL AND F I D G E
Studies were performed using both neat plasma and purified substrate. Plasma was rendered angiotensinase-free as described for substrate as.say. Prior to kinetic studies, purified substrate preparations were dialysed for 18 h at 4° against 5 I of 0-lM-sodium phosphate— 0-075M-sodiuin chloride buffer pll 7-5 containing 1 mM EDTA. This same buffer was used for dilution of substrate and renin. Gomplete survival of reactants and the end product was confirmed during all incubations. Serial dilntions of substrate without added renin were also studied as controls for the presence of endogenous renin.
RESULTS. Renin substrate in neat plasma. Expressed us angiotensin II content, the ineiin substrate level in normal sheep was 0-41 ± 0 03 fig/ml {± SE, n ^ 5) rising to 2-45 ± 0-25 /xg/ml (± SE, n = 12) at day 6 post-nephrectoniy. Specifie aetivity with respect to total protein rose 5- to lO-fold (maxinuun 0 0 4 /xg/mg protein) in different sheep. In the 5 sheep in which it was studied there was a progressive increase in plasma substrate concentration over the 6 days following nephrectomy without evidence of a plateau being reached. Renin stibstrate concentration in pooled normal male and female human plasma was 1 jLig/m! and in pooled pregnancy plasma was 7 /ig/ml. The specific activity of the pregnancy plasma was 0-09 /ig angiotensin/mg protein. DEAE-Sephaclex chromatograpinj. From five separate columns, renin substrate in nephrectomi.sed sheep plasma eluted as a single, smooth peak between 0-lOM- and 0-15M-sodium chloride at the beginning of the albumin peak. Renin substrate from normal sheep plasma eluted in the same position. Recovery was 90-100^ and specific activity increased up to lO-fold (reaching 0-33 jUg angiotensin/mg protein) in fractions from the first half of the peak. This chromatographed substrate was free of angiote-nsinase. The elution position of angiotensinase was not determined. Human substrate also eluted as a single peak, but in contrast to sheep substrate its elution was between 0-22M- and 0 25M-sodium chloride at the end of the albumin peak. Substrate in plasma from both pregnant and non-pregnant subjects behaved identical!). Recovery was 40-50% and the increase in specific activity varied from 2- to 12-fold in different experiments. DEAE-Sephadex cohmm ehromatography was effective in producing a human substrate preparation free of angiotensinase with low endogenous renin activity. Renin eluted between 0-18M- and 0 22M-sodium chloride, in front of the albumin and the substrate peaks. The highest specific activity human substrate obtained with this system was 0-9 yxg angiotensin/mg protein. The relative elution positions of sheep and human substrates from a mixture of each is shown in Fig. 1. In such an experiment, each substrate could be identified by incubating the fractions with sheep renin which released angiotensin from only the homologous stibstrate, whereas human renin acted upon both substrates.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
81
Both chromatographed substrate.s were stable during incubation at 37° and were resistant to freezing and thawing.
FRACTION NUMBER
Fig. 1. Ehition pattern of sheep and hnnian renin substrates froiu a mixtnre of sheep and human plasma, on a DEAE-Sephadex A50 column (100 cm x 2-5 cm) e(iuilii)rate(l with 0-02M-sodium nhospluite bufTer pH 7 5 and ehited with a linear gradient of sodium chloride. Fractions of volmnt- 10-0 ml were collected. Continnous trace is the nncaiibrated al).sorbance scan at 280 nm. Sheep sulxstrate ( A ) and human substrate ( • ) elute at 0-12 aud 0'24M-sodium chloride respectively.
Gel filtration chromatography of post-DEAE-Sephadex fractions. On Sephadex C-200 httle further increase in specifie aetivity of either sheep or human snbstrate was achieved above that obtained on DEAE-Sephadex. On Sephadex G-lOO (Fig. 2), sheep but not human substrate was widcl\' separated (80 ml) from the protein peak with high recovery (90-100%). Specific activity of sheep substrate in four separate experiments reached 8, 6, 5 and 5 /ig angiotensin/mg protein. This substrate preparation was stable when stored at -15° and was not affected by thawing, but lost activity over 4 days at 4°. Human substrate did not penetrate the gel phase of C-lOO Sephadex and no further increase in specific activity was achieved. Molecular weight. On Sephadex C-100, sheep substrate behaved as a molecule smaller than ijovine serum albumin monomer with a mol. wt. of 52,000. Sephadex G-200 was required for mol. wt. estimations of human substrate. It behaved as a molecule larger than bovine sernm albumin monomer with a mol. wt. of 82,000. Suhstrate specific activity. Calculating from the estimated molecular weights and assitniing that one mole of angiotensin is released from one mole of protein substrate, the highest specifie aetivity obtained for sheep substrate was 0-4 g substrate/g protein (8 nmole/mg protein) and for huuian substrate was 0-07 g substrate/g protein (0-9 nmole/mg protein).
82
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE
Kinetics. (i) Human renin-sheep suhstrate. Figure 3 shows velocity/substrate plots for both neat plasma from nephrectomised sheep and the purified sheep substrate. The only difference between the two curves is the plateau in velocity at the high protein concentrations of neat plasma. Omitting the plateau, linear transformation of this data yielded identical K,,, values for each substrate. The mean of four separate experiments was 0 29 /xM (0-21, 0 24, 0 29, 0-40).
FRACTION NUMBER
Fig. 2. Gei filtration on a Sephadex G-lOO phosphate buffer pll the uncalibrated
of sheep substrate after prior DEAE-Sephadex column chroinatiigraphy, column (100 cm x 2'5 em) equilibrated and eluted with 0-02M-sodimn 7-5. Fractions i)f volume 10-0 Till were collected. C:ontiiiuons trace is abs()ri)auce at 280 lun. Sheep substrate ( • ) elnted after 210 nil.
RENIN SUBSTRATE CONCENTRATION
Fig. 3. Eftect of sheep renin substrate concentration ou initial velocity of reaction with human renin (8 x 10-^ Goldblatt Units/ml). Abscissa: Rate of substrate degradation where 1 nniole/l/h is equivalent to an angiotensiu II generation rate of 1 HE/Vh. Specific activity of purified suhstrate ( • ) was S rmiole/mg protein and for neat nephrectomy plasma ( O ) 0-05 iimole/ing protein.
SPECIES DIFFERENCES IN RENIN SUBSTRATE
83
(ii) Human remn-human .nibnirate. Human sulistrate from normal plasina, pregnancy plasma and from DEAE-Sephadex column chromatography all showed similar reaction kinetics (Fig. 4). The highest specific activity hnman substrate used in this study contained onl\- 0 14 /xg angiotensin/mg protein (0-14 nmole/mg) at the highest concentration of 5-6 /Ag angiotensin/ml. At this concentration, Vmax was not approached and the K,,, derived from linear transformation of the data is accordingly subject to considerable error. In separate experiments K,,, varied widely at 7 8 , 7-5, 5-0 and 2-8 /IAM (mean 5 7 fxWl). The difficulty of consistently preparing high specific activity human substrate free of endogenous renin prevented more accurate determinations.
RENIN SUBSTRATE CONCENTRATION (/.M)
Fig. 4. Effect of human and sheep renin substrate concentration nn initial reaction veloeity with human renin ( 8 x 10--I Goldljlatt Units/inl). Abscissa: Rate of substrate degradation where I nmole/f/h is equivalent to an angiotensin gc'neration rate t)f 1 n g / l / h . Lower curve; human substrate preparations. Post-DKAE-Sepliadex human substrate ( O ) - Neat pregnancy plasma ( • ) . Plasma from women takinj; oral contraceptives ( • ) . Pooled normal human plasma ( A ) . Upper cnr\e: purified sheep substrate ( • ) . Means ± SE, n = 4.
(iii) Hutnan renin in a mixture of human and sheep substrates. Hnman pregnancy plasma at the constant renin substrate concentrations of either 1-0 or 2 7 ^g angiotensin/ml was mixed with serial dilutions of sheep substrate from 3-0 down to 0 25 /ig angiotensin/ml. Initial velocity was measured both with and without the addition of 10" •' Goldblatt Units of human renin/ml of incubate. Initial velocity was also measmed in control incubates containing the same concentrations of sheep substrate and renin bnt without added human plasma. At all sheep substrate concentrations in the mixed substrate system, total reaction rate was not altered by the presence of human substrate.
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE IV Sheep renin-sheep suhstrate. Sheep sul)strate, whether as unpurified nephrectomised phisma or after purification, displayed similar reaction kinetics (Fig. 5). K,,, for both reactions was 2-0 fiM. Unpurified normal sheep plasma substrate possessed identical kinetic characteristics to those of nephrectomised sheep plasma substrate and the purified substrate. For this comparison the higher specific activity substrates were diluted with buffer until substrate concentration equalled that of normal plasma. Initial velocity with added renin was then identical in all three substrate preparations.
0
2
4
6
RENIN SUBSTRATE CONCENTRATION (^Wj
Fig. 5. Lower curve shows effect of sheep renin substrate concentration on initial reaction veloeity with sheep renin. Purified substiate (•). Neat nephreetomy plasma (A). Nonnal sheep plasma (O)- ^a, = 2 \i\\. \',,,ax = 100 nmole/I/h. Upper curve is a derived plot of ths human renin-sheep suhstrate reaction assuming a Vj^j^jj of 100 nmole/I/h and K^n of 0-29 jiM.
Formation of angiotensin from renin substrates by pepsin. Pepsin is known to act on ox and horse substrates to form angiotensin II (de Fernandez, Paladini and Deluis, 1965). Figure 6 shows that whereas pepsin (Sigma, hog pepsin, twice cr\'stallized) acts on sheep substrate at pH levels 4-0 and 6-5, no angiotensin forms from human substrate under the same conditions, Human renin continued to release the same amount of angiotensin from human substrate after prior incubations of the substrate with pepsin (Fig. 5), indicating that the human substrate was resistant to proteolysis by this enzyme. With nephrectomised sheep plasma, pepsin (2 mg/ml) formed angiotensin at all pH levels between 2 and 6-5. Above pH 7-0 no formation occurred. Below pH 4 0 loss of formed angiotensin was rapid. At pH 6-6-5 no loss of
SPECIES DIFFERENCES IN RENIN SUBSTRATE
85
either formed, or added angiotensin, occurred during 60 min incubations with pepsin, and the amount generated was not significantly different from the maximum vield with renin itself.
\ HUMAN
P E P S I N PH 5 . 0
7 HUMAN
R E N I N P H 7 . 5
R E N I N
PH 7 . 5 Fig. 6. Actioii of lioy pepsin on sheep and human renin substrates. Rat blood pressurt' assay against standard angiotensin II e.xpressed in nanograms. Nephrectomised sheep plasma (Sheep). Pregnancy plasma (Ihnnan). Concentration of pepsin, 2 nig/ml; Human renin, 0-5 Coldblatt Units/ml. Incubation time 60 min.
Human substrate in Colin fractions. Fractious IV-4, IV-5 and IV-6 (Commonwealth Serum Laboratories, Melbourne) contaiTied respectively 0 008, 0 007 and 0 /ig angiotensin/mg protein. Such low specific activities made these fractions of less use than pregnancv plasma for purification purposes. DISCUSSION. Chromatography on DEAE-Sephadex followed by Sephadex G-lOO yielded a renin substrate from nephrectomised sheep plasma with specific activity 1,200 times (8 nmole/mg protein) that of normal sheep pUusma. A specific activity equivalent to this was achieved with hog renin substrate by Skeggs et al (1963) but the procedvn-es and losses were far more extensive. At all stages of purification there was no evidence for heterogeneity of sheep substrate as reported for hog substrate (Skeggs ct al, 1963) and supported iu preliminary studies by Dahlheim et al (1969). Reaction kinetics of sheep renin reacting with purified or unpurified sheep substrates, whether from normal or nephrectomised animals were not appreciablv different. This suggests identity of substrates. The K,,, for the homologous reaction in sheep (2 0/iM) was higher than for the heterologons humau renin.sheep substrate reactiou (0-29 ^M) indicating a much higher affinity between the latter reaetants. This property has been exploited in the development of a human plasma renin assay using sheep substrate where zero-order kinetics are achieved (Skinner, 1967; Stockigt et al. 1971). The K,,, for the heterologous reaction lies within the range 0 1 5 to 1 3 ^M found for a variet\' of animal
86
SKINNER, DUNN, MAZZETTI, CAMPBELL AND FIDGE
substrates reacting with hnnian renin (Brown et al, 1964; Could, Skeggs and Kahn, 1966; Dahlheim, Weber and Walter, 1970; Wuldhausl and Lewandowski, 1973). Human substrate behaved very differently from sheep substrate in the same chromatographic systems. It appeared to be a more anionic and larger protein than sheep substrate. The molecular weight of 82,000 is cousiderably greater thau that of sheep (52,000) or hog (57,000) substrates (Skeggs et al, 1963). However, the validit) of the present method might be (juestioued, since, as has been shown for some glycoproteins, elution from Sephadex is not always predictable from molecular weight (Andrews, 1965). Skeggs et al (1963) have shown that hog substrate has a small carbohydrate moiety which, if considerably larger in human substrate, might account for the displacement on Sephadex. Differences from sheep substrate were also found in the resistance of hnman substrate to the actions of both pepsin and sheep renin and also the high K,,. for the humau renin-human substrate reaction. Again, such differences might be due to a large carbohydrate moiety. Difficulty was experienced in consistently preparing a human substrate of high specific activity, but the single process of DEAE-Sephadex chromatography yielded some fractions with higher purity (0-9 nmole/mg protein) than achieved in previous work (0 5 nmole/mg protein; Rosenthal et al, 1971). Difficulty in purifviug humau substiate prevented accurate kinetic studies being performed bv conventional methods since V,,,,u was not approached. The mean K^ value of 5 7 jLiM contrasts with such values as 018.5 /iM (Haas and Coldblatt, 1967), 0-07/iM (Poulsen, 1968), 0155/iM (Roseuthal et al, 1971), 0-931 fxW (Could and Creen, 1971), 1-1 /xM (Krakoff, 1973) and 0-4 fxM (Favre and Vallotton, 1973) obtained in studies iu whicli concentration and specific activity of substrate were lower than in the present work. High values are, however, apparent in the publications of Helmer and Judson (1967), Skinner et al (1969, 1972) and Waldhansl and Lewandowski (1973). Skeggs et al (1968) described K,,, values between 3 6 and 50 /xM for a series of short linear synthetic peptide substrates reacting with humau renin but their findings do not assist in understanding the basis for the low affinity between the human reactants, because, for native substrates, affinity is likely to depend to some extent upon protein structure at a distance from the hydrolytic site. Studied at pH 7 4, K,,, values for the homologous reaction in rats (2-4 /iM; Oith et «/„ 1971) and rabbits (2 3 /xM; Campbell et al, 1973) are similar to the present finding for sheep (2 0 fiM). All of these values are considerably less than for the human and raise the possibility that a low affinity renin substrate might offer some evolutionary advantage for man. The finding that in a two substrate system the presence of human substrate did uot reduce reactiou rate between hnman renin and sheep substrate has also been reported by Stockigt et al (1971). In the presence of marked differences in initial velocities with each substrate, failure of hnman suhstrate to reduce reaction velocity in a two substrate system confirms that affinity between the homologous reactants is relatively low. The further possibility, tliat V,,,,,, with
SPECIES DIFFERENCES IN RENIN SUBSTRATE
87
both sheep and humau substrates is the same, could only be tested with purer preparations of human substrate. If this proves to be so, K,,, for the human reaetants could be accurately calculated from the data in Fig. 4, using the Michaelis equation. A value of 7 3 (xW is then predicted. To date the wide range of K,,, values reported for the human reaotants has precluded general agreement on the influence of substrate concentration on angiotensin production in vivo. Differences in the conditions of incubation or assay methods arc not obviously the cause of this and other factors are possible. The problem is exemplified by the recent report of Favre and Vallotton (1973) that affinity of humau renin for renin substrate varies inversely with reaction velocity. Such a fiuding defies any simple explanation including that offered by the authors of the presence of a competitive inhibitor in the plasma snbstrate preparation. No snch anomalies were seen in the present stndy and we conclude that in sheep and man the normal concentration of plasma substrate is at or near the first order range. Substrate concentration in vivo is therefore as important as enzyme concentration in determining production rate of angiotensin. In man it has been established that substrate levels increase in severe hvpertension (Conld and Creen, 1971), duriug pregnancy (Helmer and judson, 1967) and with oral contraceptives (Skinner et al, 1969). Indeed, renin substrate coucentration in al! species thus far studied (BUujuier, 1965; Ryan and McKenzie, 1968) is in a position to exercise control over angiotensin productiou and must therefore be considered an important determinant of circulator)- homeostasis. Acknowledgements. We (.-xprcs.s our thaiiks to Dr. Margaret Smith of the University Department of Obstetrics and Gynaecology. Royal Women's Hospital, Melljourne, for assistance with the collection of human plasma samples, and also to Dr. D. Denton and to the late Dr. J. Goding for providing facilitie.s for the collection of sheep blood. This work was supported l)y a grant from the National Health and Medical Research Council of Australia, REFERENCES. ANDHEWS, P. (1964): 'Estimation of the molecular weights of proteins by Sephadex gel-filtration.' Biochem. /., 9t, 222. ANDREWS. P. (1965): 'The gel-filtration of proteins related to their molecular weights over a wide range.' Biochem. /., 96, 595. , P. (1965): 'Kinetic studies on renin-angiotensinogen reaction after nephrectomy.' Am. } . PhysioL. 208, 1083. BROWN, J. J.. DAVIES, D . L., LEVER. A. F., ROBERTSON, J. I. S., and TREE, M .
(1964): 'E.stiniation of renin in human plasma.' Biochem. J., 93, 594. CAMPBELL,
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S.
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(1971): 'Measurement of renin concentration using i-'"'I-labelled sheep renin substrate.' Aiist. N.Z. } . Mad. 2. 102 p.
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DAY, A. J. (1973): 'Cellophane perinephritis hypertension and its reversal in rabbits.' Circulation Re.s.. 33, 105. DAHLHEIM, H.. WEBEH, P.. and
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O. (1970): 'Comparative study of enzyme kinetics on standardised human renin with four heterolojious purified renin substrates.' VpAgers Arch.. 317, 344. DAHLHEIM. \\., WEUEH, P., HAROLD, I.. PETSCHAUEH, K., and TIIUHAU, K. (1969):
'Preparative methods to purify renin substrate.' In "Progress in Nephrology", C. Peters and F. Roch-Raniel (editors), Springer-Verlag. p. 337. DE FERNANDEZ. M. T. F., PALADINI, A.
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and DELUIS, A. E. (1965): 'Isolation and identifieation of a pepsitensin.' Bioclicm. /., 97. 540..
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WEBB,
(1964):
BOSENTHAL, J., WoLFF. H. P.. WEBER, P.,
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E . C.
"Enzymes", 2nd edition, London, p. 10.
FAVRE, L.. and VALLOTTON. M . B . (1973):
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