Evidence for the glycoprotein nature of kidney renin

Biochimica el Bioph)'Mca Acta, 494 (1977) 162 171 .!'~, Elsevier/North-Holland Biomedical Press BBA 37750

EVIDENCE FOR THE G L Y C O P R O T E I N N A T U R E OF KIDNEY REN1N

MORTON P. PR1NTZ and ROBERT T. DWORSCHACK

Division of Pharmacology, M-OI3, Department ~[" Medicine, University 0[' Cal(Jarnia, San Diego, La Jolla, Calif. 92093 (U.S.A.) (Received March 7th, 1977) (Revised manuscript received May 20th, 1977)

SUMMARY

The glycoprotein nature of renin isolated from either rabbit or human kidney has been demonstrated by affinity chromatography on concanavalin A-Sepharose. The bulk of rabbit renin activity bound to concanavalin A is released by 20 to 50 mM a-methyl-D-mannoside. Adsorption of renin is prevented by periodate oxidation prior to chromatography. Mild acid treatment (pH 2.5) prior to chromatography does not alter the concanavalin A binding profile although the pl values of native rabbit renin (5.1-5.6) are shifted into a broader distribution (4.7-6.4). The molecular weight values of rabbit renin obtained by gel filtration and those from zone centrifugation are identical (37 000 -5 I000), consistent with a low percent of carbohydrate in the glycoprotein. A hydrophobic contribution to the binding of renin by concanavalin A is evident since, in the presence of mM Ca z+ and Mn 2+, higher concentrations of c~-methyl-D-mannoside are required to affect the same release of renin at 23 °C compared to that at 4 c'C. Furthermore, 25 ~ ethylene glycol releases renin in the absence of (t-methyl-D-mannoside. It is concluded that renin contains a small number of carbohydrate residues in relatively close proximity to a hydrophobic surface which enhances the interaction with concanavalin A.

INTRODUCTION

Renin (EC 3.4.99.19) is a highly specific acid protease which catalyzes the hydrolysis of a leucyl-leucine bond in angiotensinogen, the a-globulin prohormone of the angiotensin peptides, and thereby liberates the decapeptide angiotensin ! [1]. Renin is released into the circulation from the kidney where it is synthesized and stored in secretory granules [2, 3]. The storage form is probably a proenzyme [4-6] which is activated either prior to or following secretion [6, 7]. Multiple forms of renin activity can be differentiated on the basis of molecular weight or isoelectric point [8, 9]. Although isoelectric point heterogeneity is a common finding with glycoproteins [10], this does not in itself constitute evidence for the presence of carbohydrate groups in renin. In the absence of highly purified enzyme suitable for carbohydrate analysis we have investigated whether kidney renin is a glycoprotein using the technique of concanavalin A-Sepharose affinity chromatography [11, 12].

163 Further, we have examined the effect of acid activation, periodate oxidation, buffer composition and temperature on this binding. Lastly, we have compared the molecular weights of renin obtained by gel filtration and zone centrifugation. MATERIALS AND METHODS

Rabbit and human renin. Partially purified human kidney renin (spec. act. 0.13 WHO units/mg protein)* was kindly supplied by Dr. Erwin Haas and was used without further purification. Rabbit renin was isolated by the methods of Ryan et a~. [13] from frozen New Zealand white-rabbit kidneys. The kidneys, stored at --60 °C, were partially thawed and homogenized in a volume of cold distilled water equal to one-half their weight. The homogenate was stirred for 1 h at 5 °C, diluted with 10 volumes of distilled water and filtered through cheesecloth. Lipids in the filtrate were extracted with 0.1 volumes of cold toluene and the aqueous phase centrifuged at 27 000 × g for 30 rain. Renin activity in the supernatant was precipitated by the slow addition of solid ammonium sulfate (Schwarz/Mann, enzyme grade) to a concentration of 40 ~o (w/v). After centrifugation, the pellet was resuspended in 10 mM EDTA, pH 6.0, and dialyzed at 5 °C for 36 h against three changes of the same buffer. Insoluble protein was removed by centrifugation and the supernatant stored at - 1 2 °C. This preparation was used as the starting material for most of the studies reported below. We frequently found that such a preparation would initially contain only low levels of renin activity. However, if the enzyme solution was diluted 10-fold prior to assay or was stored at 12 '~C for several months, a marked increase in renin activity occurred. A similar process, cryoactivation of the proenzyme form of renin, has recently been reported by Sealey et al. [14]. Portions of the crude renin solution were further purified by isoelectric focusing in a 2 ~ ampholytes gradient (pH 4-6) using a 440 ml focusing column (LKB 8101) following procedures already reported [15]. Acid treatment. Rabbit renin solutions were acid activated by lowering the pH to 2.5 with cold 1 N sulfuric acid. After standing for l h at room temperature the solution was dialyzed for 24 h against several changes of 50 mM sodium phosphate buffer, pH 7.0, at 5 °C. Insoluble material was removed by centrifugation at 10 000 × g. The human renin preparation had already undergone acid treatment during the isolation [16] and was therefore presumed to be acid activated. Assay ofrenin activity. Enzyme activity was measured by incubating an aliquot of renin at pH 6.0 with 0.16 /~M hog renin substrate (Miles-Pentex) at 37 °C for 1-3 h. The incubation solution contained 60 mM sodium phosphate, 70 mM phenylmercuric acetate (to inhibit bacterial growth) and 15 mM EDTA. The quantity of angiotensin I generated during the incubation was determined by radioimmunoassay as previously described [15, 17] and renin activity was defined in terms of angiotensin I liberated/ml per h. " Since renin is an impure enzyme preparation, renin activity has traditionally been expressed m terms of a "Goldblatt" unit which was derived from a blood pressure bioassay. In order to facilitate comparison of data from different laboratories which utilize radioimmunoassay procedures to quantify angiotensin 1, a renin standard was prepared and distributed by the World Health Organization. The activity of this preparation is expressed in terms of WHO units which are equivalent to the original Goldblatt unit.

164

Concanavalin A-Sepharose chromatography. The binding of renin to concanavalin A-Sepharose (Pharmacia) was investigated in two buffer systems: buffer "A'" 100 mM sodium acetate, pH 6.0, containing 100 mM sodium chloride, 1 mM calcium and manganese chlorides, or buffer "B" containing only 20 mM sodium phosphate, pH 6.7. Prior to loading onto the column renin was dialyzed for 4 h against two changes of distilled water to remove any EDTA or sucrose remaining from the isoelectric focusing. The sample was adjusted to the composition of buffer A using a 10-fold concentrated buffer stock added immediately prior to addition to the column. Eluant fractions were collected from the column (1 ;~ l0 cm bed volume) into test tubes containing 1/10 fraction volume of 100 mM EDTA, pH 6.0. The latter procedures were not utilized for chromatography of renin in column buffer B. Renin activity was generally eluted from the column with a linear gradient up to 1 M ct-methyl-D-mannoside (Sigma, 99~o~ pure). The concentration of mannoside was determined by the anthrone method [I 8]. Molecular weight determination. The apparent molecular weights of native and acid-treated renins were determined by gel filtration [19]. An aliquot (2 ml) of the crude renin preparation was chromatographed at 5 :C on a G-150 Sephadex (Pharmacia) column (2.5 ~ 30 cm) at a flow rate of 15 ml/h. The developing buffer was 50 mM sodium phosphate, pH 7.0, containing 200 mM sodium chloride. The protein standards used to calibrate the column were ribonuclease, chymotrypsinogen, ovalbumin and aldolase. The method of zone centrifugation was also used to determine the molecular weights of native renin. Three linear gradients ( 5 - 2 0 ~ sucrose in 60 mM sodium phosphate buffer, pH 6.0, containing 15 mM EDTA) were centrifuged in the SW 50.1 rotor in the Beckman L2-65B at 38 600 rev./min for 9 h at 2 ~-~C. Molecular weights were calculated by the method of Martin and Ames [20] using human carbonic anhydrase (M~ = 31 000) and firefly luciferase (M~ 50 000) as internal standards. Luciferase activity was assayed by the method of McElroy and Seliger [21] and carbonic anhydrase by the hydrolysis of p-nitrophenylacetate [22]. Proteolytic activity in the renin sample did not interfere with the luciferase standard since the position of the luciferase was identical in the presence or absence of added renin. The position of the carbonic anhydrase was determined in a separate gradient tube without renin due to the esterase activity present in the partially purified preparation. RESULTS

Chromatograph)' on concanavalin A-Sepharose. The major forms of human renin bind to concanavalin A-Sepharose and are released by a-methyl-D-mannoside (Fig. l) in a manner suggesting a broad spectrum of affinities toward concanavalin A. Elution of native rabbit renin with a linear gradient of mannoside also exhibits (Fig. 2A) a wide range of affinities with the bulk of the activity desorbing at 20-50 mM mannoside. Rabbit renin will not adsorb to Sepharose 4B indicating that an interaction with concanavalin A is essential for chromatography. Pools of early and late eluting rabbit renin activities were concentrated to < 4 ml and rechromatographed ~n concanavalin A-Sepharose to determine whether these activities represent distinctly different forms of renin. The enzyme used for this study had been purified through isoelectric focusing since the crude preparation becomes unstable after a single

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ELUTION VOLUME Fig. I. Elution of human kidney renin from a concanavalin A-Sepharose column at 4 "C. The renin solution (1 ml or 1 WHO standard unit) was flowed thro'agh a 1.7 ml bed volume column and buffer A (100 mM sodium acetate, 100 mM sodium chloride, I mM each calcium and manganese chlorides, pH 6.0) was used for equilibration and development. Renin was eluted by stepwise addition of increasing concentrations of ¢~-methyl mannoside. Renin activity is expressed in terms of the rate of angiotensin I generation upon incubation with hog renin substrate at pH 6.0 and 37 °C. For further details see test. passage through concanavalin A-Sepharose. Upon rechromatography the elution p r o f i l e s o b t a i n e d f o r b o t h e a r l y a n d l a t e e l u t i n g f r a c t i o n s were s i m i l a r ; t h e a p p a r e n t m u l t i p l e f o r m s o f r e n i n all c h r o m a t o g r a p h e d in t h e e a r l y e l u t i n g f r a c t i o n . F u r t h e r s t u d i e s d e t e r m i n e d t h a t t h e o b s e r v e d l a t e - e l u t i n g f r a c t i o n s were n o t r e l a t e d s i m p l y I

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Fig. 2. Elution of native- and acid-treated rabbit kidney renin from concanavalin A-Sepharose at 4 "C. Renin activity was eluted with a gradient (0 500 mM) tz-methyl mannoside in buffer A. The column bed volume was 8 ml and the flow rate was 15 ml/h. Samples (20 ml) of either (A) native or (B) acid-treated renin supernatant were passed through the column. The total protein load was 600 mg for (A) and 80 mg for (B). 3he large enhancement in renin activity in (B) is the result of acid treatment.

166 to excess protein loading but rather the trailing occurs when large sample volumes (10 ml and greater) are applied to the columns. Acid t r e a t m e n t o f r a b b i t renin increased activity in the crude p r e p a r a t i o n 3-fold, however, the elution pattern o f acid-activated renin from concanavalin ASepharose was not significantly altered (Fig. 2B). The a p p a r e n t isoelectric points o f renin obtained by isoelectric focusing were altered by acid activation. The p a t t e r n for native enzyme (Fig. 3A) indicates that renin activity exhibits a spread o f isoelectric points from p H 5.1 to 5.6, whereas that for the acid-treated renin (Fig. 3B) had p! values ranging from 4.68 to 6.4. There is a l m o s t a 50-50 redistribution o f the renin to higher or lower isoelectric points following acid treatment. 1

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Fig. 3. lsoelectric focusing of rabbit renin in a 2% ampholyte gradient (pH 3.5-7) and I I0 ml focusing column (LKB 8101 ). (A), Native rabbit renin (2 ml or 80 mg protein) exhibited major bands of activity at pH 5.3, 5 42 and 5.5 and a minor band at 5. I. (B), Acid-treated rabbit renin (8 ml or 32 nag protein) gave major bands at pH 4.7, 5.45, 5.8 and 6.0 and minor bands at 6.2 and 6.4. The gradient was eluted at a flow rate of I. 1 ml/min (LKB perpex pump), tubes immediately stoppered and the pH measured at room temperature. Periodate o x i d a t i o n would be expected to m a r k e d l y alter those c a r b o h y d r a t e residues o f glycoproteins generally responsible for specific interactions with concanavalin A. Further, Stahl et al. [23] have reported a m a r k e d l y reduced binding o f /3-glucuronidase to concanavalin A following periodate oxidation. To evaluate this effect on renin, solutions o f the crude enzyme p r e p a r a t i o n ( p H adjusted either to 4.9 or 6.0) were incubated at 0 ~C in the d a r k for varying periods o f time with 10 m M s o d i u m periodate. C o n t r o l incubations o f the renin solutions (lacking periodate) were h a n d l e d in an identical m a n n e r to exclude effects from c o n t a m i n a t i n g proteases. The reactions at pH 4.9 and 6.0 were terminated by dilution with 50 m M sodium p h o s p h a t e buffer containing 200 m M s o d i u m chloride (at the respective p H ) followed by dialysis against the same buffer for 18 h. Aliquots o f the oxidized and control

167 renin incubations were applied to 1 ml columns o f concanavalin A - S e p h a r o s e and eluted with buffer A. The binding o f renin was m a r k e d l y decreased following o x i d a t i o n at either pH. The effect at pH 6 is clearly evident in Fig. 4. O x i d a t i o n at p H 4.9 causes a similar loss in lectin binding with c o m p a r a b l e kinetics. Renin activity was not completely stable to p e r i o d a t e oxidation. F o r example, at p H 6.0, 2 0 - 3 5 % o f the total activity was lost following 7.5 h o f incubation, whereas at pH 4.9, 35-50 °//o o f the activity was lost in 6.3 h.

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Fig. 4. Effect of sodium periodate oxidation at pH 6.0 on the binding of renin by concanavalin ASepharose. The conditions of the periodate oxidation are described in the text. Periodate-modified renin (200/d) was applied to 1 ml columns of concanavalin A-Sepharose equilibrated with Buffer A. After eluting unbound renin activity with 8 ml of Buffer A, bound activity was released with 1 M (~-methyI-D-mannoside. Percentages refer to the peak activity in relation to the total recovered activity alter chromatography. In a similar experiment at pH 4.9, after 3.8 h of periodate oxidation, 67 % of the renin failed to bind to the lectin and after 6.7 h, over 77% was nonadsorbed. Buffer c o m p o s i t i o n and t e m p e r a t u r e variation have a significant effect on the elution o f renin from c o n c a n a v a l i n A-Sepharose. Renin activity could be released at 4 C in the absence o f m a n n o s i d e with buffer A c o n t a i n i n g 25 ~ (final concentration) ethylene glycol. The total activity recovered was only 30~,, o f that following elution with 800 m M m a n n o s i d e . This diminished activity may be the result o f both less efficient elution by ethylene glycol a n d inhibition o f the renin-angiotensinogen reaction since 50°/j,o inhibition was observed with 2 5 ~ v/v ethylene glycol. Elevation o f the t e m p e r a t u r e o f c h r o m a t o g r a p h y had a p r o f o u n d stabilizing

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Fig. 5. Elution of rabbit kidney renin from concanavalin A-Sepharose at 23 ~C in buffer A. An aliquot (I ml) of native renin was applied to the column and eluted with a gradient (0-1.0 M) u methyl-o-mannoside at a flow rate of 20 ml/h. The arrow denotes the start of the gradient. effect on the interaction between the enzyme a n d the lectin. F o r example, at 23 C renin was eluted at 250 m M m a n n o s i d e in buffer A (Fig. 5), a c o n c e n t r a t i o n 5 to 12fold higher than that needed at 4 °C ( c o m p a r e with Fig. 2A). The yield o f activity eluting from the column at this higher t e m p e r a t u r e was c o m p a r a b l e to that at 4 °C. This e n h a n c e m e n t o f the a p p a r e n t binding c o n s t a n t was n o t observed if the column was equilibrated with buffer B, 20 m M sodium phosphate, pH 6.7. In buffer B the m a j o r p o r t i o n o f renin activity was eluted with 40-50 m M m a n n o s i d e at both 4-' and 23 °C. In addition, the yield o f renin activity recovered at 23 °C was a p p r o x i mately 225 % o f the recovery at 4 °C. A greater recovery o f enzyme activity u p o n elution from c o n c a n a v a l i n A - S e p h a r o s e at 23 °C with neutral p h o s p h a t e buffer has also been observed by N o r d e n and O'Brien [24] for h u m a n liver fl-galactosidase. Comparison of molecular weight determinations of renin. The a p p a r e n t m o l e c u l a r weight o f native r a b b i t renin was determined by gel filtration. In agreement with previous findings [2, 4] the m a j o r p e a k o f renin activity h a d a molecular weight of 36 500, while a lesser a m o u n t o f activity was distributed in the range o f 100 000. F o l l o w i n g acid t r e a t m e n t r a b b i t renin exhibited only a single p e a k o f activity with a m o l e c u l a r weight o f 38 000. Z o n e centrifugation o f native renin (Fig. 6) indicated a

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169 heterogenous distribution of molecular weights. While the major peak of activity centreed around a molecular weight of 36 800, larger forms of renin were observed with molecular weights ranging up to 100 000. DISCUSSION The application of concanavalin A-Sepharose for affinity chromatography of glycoproteins is predicated on the specificity of the interactions of the lectin with the polysaccharide moieties of the proteins. Extensive studies by Goldstein and associates have established that a-r~-mannopyranose and ~-r)-glucopyranose exhibit highest specificity for the saccharide binding site on the lectin [11, 12, 25]. It is also clear that these residues may occupy either terminal or interior positions in the polysaccharide and still exhibit high affinity interactions [26]. Further, Kornfeld and Ferris [26] have presented evidence that the saccharide-binding surface ofconcanavalin A can accommodate a variety of polysaccharide side chains at the same time. In addition to the saccharide binding site a hydrophobic site appears to be in relatively close proximity (35 A) on the surface of the protein [27, 28]. While the original concept that the saccharide and hydrophobic sites are coincident is no longer tenable with recent data, their relative contributions in facilitating the binding of glycoproteins is not yet fully defined. The hydrophobic component is still considered to be a non-specific site, yet there is no cogent reason why it should not make a significant contribution to the binding energy of glycoproteins and therefore be an essential part of the primary interaction. Studies by Davey et al. [29] have demonstrated the importance of this hydrophobic component in the binding of interferon to concanavalin A. "therefore, it seems appropriate to recognize that together they present the opportunity for a spectrum of binding strengths for a glycoprotein. Binding would be determined by the structure and sequence of the polysaccharide side chains and by their environment on the glycoprotein. A glycoprotein which lacks sufficient non-polar amino acid side chains in an appropriate configuration with the polysaccharide chain would therefore bind 'weakly' without any evidence of a hydrophobic component. The converse would lead to a strong interaction with both binding surfaces on concanavalin A as defined by the concentration of glycoside needed for displacement in the presence or absence of added less polar solvents. The data presented above indicate that renin exhibits properties consistent with a glycoprotein. In particular, native and acid-treated renin can be displaced from concanavalin A-Sepharose by relatively high concentrations of ¢~-methyl-r)mannoside. Further, periodate oxidation at either pH 4.9 or 6.0 markedly diminished binding of renin to concanavalin A. It is unlikely that this effect was a result of oxidation of amino acid side chains or overoxidation of residual carbohydrate fragments since these reactions are generally not significant at either pH used here [30]. There also appears to be a major hydrophobic contribution to the interaction between renin and concanavalin A. The effectiveness of ethylene glycol elution is suggestive of the presence of a hydrophobic component to the binding but does not prove it since ethylene glycol could alter the conformation or subunit interactions of the lectin. However, the increased affinity of renin for the lectin column at elevated temperature is a more positive indication of a hydrophobic contribution. Divalent metal ions may be involved in maintaining concanavalin A in a structure which

170 facilitates this interaction since tighter binding at higher temperature is not observed in the absence of the metal. There is no evidence at the present time that renin binds divalent metal ions. We have no exact information as to the carbohydrate content of renin. Indirect evidence indicates that it makes a relatively minor contribution to the total mass of the protein since the apparent molecular weights as determined by zone centrifugation and gel filtration are quite similar. Thus, the well recognized disparity between the results of these two techniques when applied to glycoproteins [20] is not evident here. In addition, preliminary studies of the effects of neuraminidase digestion of renin fail to provide evidence for the presence of sialic acid on the enzyme (Dworschack, R. T., unpublished observations). These questions will be resolved when highly purified renin is available. Acid and trypsin activation of renin converts a low activity, high molecular weight form (a proenzyme) to the major low molecular weight enzyme. It has recently been shown that if renin is isolated in the presence of several protease inhibitors the proenzyme is the predominant form obtained [31]. Despite a significant change in the apparent isoelectric point distribution following acid treatment of native renin there is no alteration in the binding to concanavalin A. The mild acid treatment would not be expected to decrease the carbohydrate content; higher temperature and longer incubation times are required to remove even sialic acid, a very labile carbohydrate residue [32]. Since acid activation appears to involve the release of a 13 000 molecular weight inhibitory fragment [6] our results indicate that the concanavalin A binding saccharide(s) on renin are retained on the 37 000 molecular weight fragment. in conclusion, our data indicates that kidney renin is a glycoprotein. However, the role of this carbohydrate in the in vivo function of the enzyme is unclear. Although glycosylation does not appear to be a requirement for the secretion of some extracellular proteins [33], carbohydrate composition does play a role in the regulation of glycoprotein turnover and compartmentalization [34, 35]. Further studies are clearly necessary to define the importance of the carbohydrate nature of kidney renin to its kinetics of reaction with the protein substrate and to its physiological functions. ACKNOWLEDGEMENTS We wish to thank Dr. Erwin Haas for the generous supply of human kidney renin, Dr. Marlene DeLuca for advising in the measurement of firefly luciferase, Dr. Palmer Taylor for advise in the assay of carbonic anhydrase and Dr. Russell Frost for valuable discussions on the periodate oxidation of glycoproteins. In addition we wish to thank Dr. D. R. Bangham, Medical Research Council, and the World Health Organization for the samples of the WHO renin standard. This work was supported by a grant from the National institutes of Health, Grant HL 15808, and by an award from the San Diego County Heart Association.

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171 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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