Biomedical Research 2 (5) 552-559, 1981

DIRECT EVIDENCE, USING Pro-Phe-ArgCH2Cl, THAT PLASMA KALLIKREIN HAS A ROLE IN ACID ACTIVATION OF INACTIVE RENIN IN PLASMA FROM NORMAL SUBJECTS

BRIAN J. MORRIS and JOSEPH G. McGIRR Department of Physiology, The University of Sydney, New South Wales, 2006, Australia

ABSTRACT A role for plasma kallikrein in the activation of inactive renin elicited by acidification of human plasma was investigated using the new highly selective inactivator of plasma kallikrein, Pro-Phe-ArgCH,Cl, as a probe for this enzyme activity. Pro- Phe-ArgCH,Cl, tested over the concentration range of 10"“ to 10"‘ M, produced virtually identical inhibition of plasma kallikrein activity and of renin activity seen after activation of inactive renin by treatment of plasma to pH 3.3 for 24 hr at 4°C and then to pH 7.4 for 24 hr at 4°C. The concentration needed for 50% inhibition was 1.3 >< 10-6 M. Hill plots demonstrated a 1 : l molecular interaction. Pro-Phe- ArgCH,,Cl had no effect on the activity of renin itself so that the reduction in renin levels seen after activation was presumably due to an action of the inhibitor on a factor having a role in acid activation of inactive renin. The identical effect on renin levels after activation and plasma kallikrein activity indicated that plasma kallikrein was this factor. Furthermore, plasma kallikrein and inactive-renin- activating activity co-eluted when pre-acidified plasma was run on Sephacryl S-200. Moreover, the latter was inhibited completely by Pro-Phe-ArgCH2Cl. The way in which plasma kallikrein works in acid activation need not be by a direct action on inactive renin to activate it. The present results nevertheless provide compelling evidence for the identity of plasma kallikrein as the serine proteinase with a key role in the phenomenon of acid activation of inactive renin in plasma from normal subjects.

KEY WORDS inactive renin / plasma kallikrein / human plasma

Human plasma contains a large reservoir of teinases, viz. aprotinin (11, 20, 21, 40), soybean potential renin activity (‘inactive renin’). How- trypsin inhibitor (1, 12, 20, 21), di-isopropyl- ever, it is not known whether this reservoir is phosphorofiuoridate (1, 2, 12, 40) and benza- ever tapped in viva. In virro inactive renin can midine (1), it was proposed that serine protein- be revealed by acidification to pH 3.3 for 24 hr ases play a role in activation by acid or cold. at 4°C followed by 24 hr at pH 7.4 and 4°C (6, These findings initiated a search for the 22, 25, 38, 41, 46) or by incubation for several particular serine proteinase(s) responsible. days at —4°C (1, 2, 12, 40) or by incubation with Studies of plasma deficient in plasma prekalli- a variety of proteinases (5, 6, 8-10, 14, 15, 19, krein (Fletcher trait) have demonstrated abnor- 28-30, 33--39, 41, 43, 45, 50). Since, in plasma, mally high inactive and low active renin levels, both acid activation (1, 2, ll, 20, 21) and cryo- as well as impaired acid activation (7, 44). activation (1, 2, 12, 40) appeared to be inhibited After performing a number of elegant experi- substantially by general inhibitors of serine pro- ments with the plasma from such patients good PLASMA KALLIKREIN AND INACTIVE RENIN 553

evidence was produced that plasma kallikrein is Renftz Assay necessary for acid activation (7, 44). However, in a recent report, cryoactivation was neither Renin was measured by incubating 50 /11 of impaired in Fletcher plasma nor inhibited by sample at 37°C for 3 hr with 200 /11 of nephrec- soybean or lima bean trypsin inhibitors (49). tomized sheep plasma buffered to pH 7.4 with Thus plasma kallikrein may not be involved in 50 mM sodium phosphate and containing >10 cryoactivation in Fletcher plasma, even though >

Fletcher patients may be complicated by the national Reference Preparation of Human Renin E E 2 existence in such plasma of differences in other (code 68/356; National Institute for Biological E l enzymatic pathways besides those associated Standards and Control, Holly Hill, London) was s i E with a mere prekallikrein deficiency. found to generate 75,800;I;8,800 SD pmol angio- s i In the present study we used a new selective tensin I-hr"1/ml (n:8). 2 inhibitor of plasma kallikrein, Pro-Phe-Arg- i CHZCI (16, 17), and plasma from normal indi- E viduals. The results provide the first direct Plasma Kallila'ez'n Assay proof of a role of plasma kallikrein in acid acti- Plasma kallikrein was measured by its amidolytic vation of inactive renin in normal human plasma. activity at 4°C. This was done by monitoring Some of this work was reported recently in a the increase in absorbance at 405 nm in a 2.5 ml brief preliminary communication (27). mixture containing 100 /Jl plasma sample, 0.8 >< 10"4 M chromozym PK (N-Bz-Pro-Phe-Arg-p- MATERIALS AND METHODS nitroaniline; Boehringer Mannheim), 8 mM 6- caproic acid, 35 mM Tris, pH 7.9, 132 mM ll/{aterials NaCl and 9 mM EDTA (26). AA40,/min was Fresh citrated plasma was obtained from the converted to katals/ml (:amount of enzyme Sydney Blood Bank and pooled samples, each that hydrolyses 1 mol substrate per sec) assuming from 60 subjects, were stored at —20°C. Pro- an extinction coefficient of 10,600 M"1 cm"1 Phe-ArgCH2Cl was a kind gift from Dr Charles (26) and also to mU/ml (47). Kettner and Dr Elliott Shaw, Brookhaven National Laboratory, Upton, NY, and was stored as a 10"?‘ M solution in 1mM HCl at Gel Flltration —20°C. Gel filtration was performed using a 1.5 >< 90 cm column of Sephacryl S-200 (Pharmacia) equili- brated with 10 mM sodium phosphate buffer, Treatment of Plasma pH 7.4, containing 200 mM NaCl and 3mM Plasma was treated according to a conventional NaN3 at 4°C. Plasma was preacidified to pH acid-activation procedure, viz. dialysis for 24 hr 1.5, 3.3 or 4.8 for 24 hr at 4°C, then dialysed in at 4°C in 50 mM glycine-HCl buffer, pH 3.3, pH 7.4 buffer overnight and 2 ml was applied to containing 100 mM NaCl and 3 mM Na2EDTA, the column. Fractions of approximately 2.8 ml followed by restoration of the pH to 7.4 with were collected at a flow rate of 0.2 ml/min. 1/30 volume of 2 M NaOH and 1/12 volume of Elution volumes were determined by weighing 4 M Tris buffer, pH 7.4, and incubation for a each fraction. Protein in the fractions was further 24 hr at 4°C (25, 43). To test the effect monitored by the absorbance at 280 nm. Plas- of the selective plasma kallikrein inhibitor, Pro- ma kallikrein activity of fractions was deter- Phe-ArgCH2Cl was added to samples at the mined spectrophotometrically over 3 hr as time of adjustment of pH from 3.3 to 7.4. The described above. Inactive-renin-activatin g activ- final concentrations of Pro-Phe-ArgCH2Cl in ity was measured by incubating 0.5 ml fraction plasma samples were 10‘3, 10“7, l0"6, 10"5 and with 0.5 ml human amniotic fluid at 25°C for 60 10"‘ M. hr and determining the increase in renin. As 554 B. J. MORRIS and J. G. McGIRR

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L ,-_,_, 1 l A LL11, L,,,,,,Ll O 10 20 O 10 20 Time of incubation Time of incubation of pre-acidified plasma of 18h samples with at pH7.4,4°C (h) chromozym PK (min) Fig. 1 Effect of Pro-Phe-ArgCH2Cl at concentrations of 0 (O), 10-8 (O), 10"’? (A), 10"5 (A), 10‘5 (I) and l0‘4 M (El) on the generation of renin activity in the alkaline phase of acid activation (left) and the enzymatic activity of plasma kallikrein (right). well, this was done in the presence of Pro-Phe- 95 i8 pU/ml and Fig. 1 demonstrates the fur- ArgCH2Cl, 10“6 M final concentration, and ther increase over 24 hr when the pH was then also with a mixture of phenylmethanesulfonyl adjusted to 7.4. Pro-Phe-ArgCH2Cl caused fluoride, 5 mM, and soybean trypsin inhibitor, concentration-dependent reduction in the renin 0.3 mg/ml. The latter mixture was added to all activity measured during this alkaline phase of samples before renin assay. Amnitoic fluid was activation. Renin levels observed in the pres- used in preference to plasma because the lower ence of 10"‘ M Pro-Phe-ArgCH2Cl, the highest concentration of proteinase inhibitors means concentration of inhibitor used, were similar to that prior acidification, which is usually required renin levels after pH 3.3-treatment alone: 971 12 to reduce the high proteinase inhibitor activity vs. 95 i8 ,uU/ml, respectively. Pro-Phe-Arg- in plasma (and which also activates prekallikrein CHZCI had no effect on the activity of renin and inactive renin), was not necessary. The itself. Since both here and in reference 7 renin column was calibrated using Blue Dextran (M, attained plateau levels at 24 hr, the 24 hr values 2,000,000), catalase (M, 210,000), bovine serum were taken and expressed as a percentage of the albumin (M, 67,000), ovalbumin (M, 45,000) and total renin activity seen in the ‘alkaline’ phase. 06-chymotrypsinogen (M, 25,000). Fig. 2 shows these results for 12 experiments, with renin plotted against concentration of Pro- RESULTS Phe-ArgCH 2C1. Plasma prekallikrein is activated by acidifica- Whole Plasma tion and plasma kallikrein activity increases with Acidification of plasma activated inactive renin. time after adjustment of the pH to 7.4, reaching Renin in unacidified plasma was 43 i8 SD a plateau at 18 hr (7). The activity attained at /1U/ml (n=12) and increased with pH 3.3—pH 18 hr at 4°C in the present experiments was 0.4 7.4 treatment to 198i12 ,uU/ml, indicating that nkatal (20 mU)/ml. With acid/kaolin activa- 78% of total renin was inactive renin. After tion (26) plasma kallikrein activity at 37°C was treatment to pH 3.3 alone renin increased to 1.0 nkatal (50 mU)/ml, a value similar to that PLASMA KALLIKREIN AND INACTIVE RENIN 555

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O__ l._,,,,,,,,, W l8_ L;_____.. _L ... _..4l ,__ .5-. , 0 10“ 10‘ 10" 10° 10'1‘ Q -1.0 Concentration of Pro—-Phe-ArgCH2Cl (M) O log [Pro—Phe—ArgCH2Cl] Fig. 2 Effect of the selective plasma kallikrein in- activator, Pro-Phe-ArgCH;;Cl, on renin measured Fig. 3 Hill plot of data taken from Fig. 2. The after dialysis in pH 3.3 buffer for 24 hr at 4°C and slope of the line was 1.0, indicating that one mole- subsequent incubation at pH 7.4 for 24 hr at 4°C cule of Pro-Phe-ArgCH2Cl was inhibiting one mole- (meaniSD, n=12) (0) and on plasma kallikrein cule of plasma kallikrein, as it was the factor having a activity, measured at 4°C in the same samples after role in acid activation of inactive renin. (Q, renin; 18 hr (mean;l;SD, n=5) (O). O plasma kallikrein). found by others (47). Concentration-dependent would have undergone during chromatography. inhibition of plasma kallikrein activity by Pro- Only partial activation of inactive renin was Phe-ArgCH2Cl was observed (Fig. 1), with 1.3 >< allowed in order for zero order conditions with 10“'6 M being required for 50% inhibition (Fig. respect to substrate (inactive renin) to be ob- 2). As shown in Fig. 2, inhibition curves for served. Gel filtration of pH 3.3-pretreated plasma kallikrein activity and activated renin plasma revealed inactive-renin-activating activ- were similar. Hill plots (24) of the data gave a ity in a similar position of elution as above. straight line of slope 1.0 for both renin and This was also true of fractions lyophilized before plasma kallikrein activities (Fig. 3), indicating incubation with amniotic fluid, although more that one molecule of Pro-Phe-ArgCH2Cl was inactive renin was activated. In plasma treated reacting with one molecule of plasma kallikrein only to pH 4.0 most of the plasma kallikrein and that one molecule of Pro-Phe-ArgCH2Cl activity emerged in the region of 0t;,-macroglo- was also reacting with one molecule of the factor bulin. In its complex with the latter plasma involved in acid activation of inactive renin. kallikrein retains its enzymatic activity against low M, substrates the size of chromozym PK. Column Fractions Gel filtration of pH 2.5-pretreated plasma at pH DISCUSSION 7.4 demonstrated a peak of plasma kallikrein The present results establish that plasma kalli- activity of approximately 130,000 M, (Fig. 4). krein has a role in conventional acid-activation Column fractions were capable of activating of inactive renin in plasma from normal subjects. inactive renin and the profile of this activity was This conclusion is based on the results of experi- almost. identical with that of plasma kallikrein ments using Pro-Phe-ArgCH2Cl which owes its activity. Moreover, the inactive-renin-activab inhibitor specificity to its resemblance to the Pro- ing activity could be inhibited not only by phenyl- Phe-Arg-Ser site in kininogen hydrolysed by methanesulfonyl fluoride and soybean trypsin plasma kallikrein (16, 17). In this respect the inhibitor, but also by the selective plasma kalli- preference of Pro-Phe-ArgCH2Cl for inhibition krein inactivator, Pro-Phe-ArgCH2Cl. The of plasma kallikrein exceeds that for plasrnin by a concentration of the latter used, 10*“ M, is factor of 48, factor X, by 107, urinary kallikrein approximately the dose expected from Fig. 1 to by 300, thrombin by 1,200 and urokinase by be just suflicient for almost complete inhibition 100,000 (16, 17). Pro-Phe-ArgCH2Cl is there- of plasma kallikrein activity considering the fore a suitable probe for testing whether or not approximately 10-fold dilution that the latter plasma kallikrein is the serine proteinase in- 556 B. J. MORRIS and J. G. McGIRR

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(ARen'n)pU/m I I renact've-- n ~---- *1 . kma D3 I Absorban O )0-o‘...... __0 P"'as Ql_ Il.:| figl H ...-Q _ . I . or LL I ill‘ n =I 5 A “ ‘ ID 2» . l. -...... l I ,1 , l 1,1 ....l L L_.__.l_.l 40 60 I 80 100 Volume of eluate (ml) Fig. 4 Gel filtration of pre-acidified plasma on Sephacryl S-200. Shown are inactive- renin-activating activity (0), plasma kallikrein activity (O) and protein (...). The basal level of active renin (200 ,u-U/ml diluted amniotic fluid) was subtracted from the total renin measured in each fraction to give the A renin values shown. The effect of Pro-Phe- ArgCH;,Cl (A) and of phenylmethylsulphonyl fluoride plus soybean trypsin inhibitor (I) on inactive-renin-activating activity is also shown. volved in acid activation of inactive renin in present results have also demonstrated a clear plasma. If a serine proteinase other than similarity in the elution from Sephacryl S-200 of plasma kallikrein had been involved then the plasma kallikrein activity and the activator of renin curve in Fig. 2 would have been displaced inactive renin. The fact that the latter could be substantially to the right of the curve for plasma inhibited by Pro-Phe-ArgCH2Cl further points kallikrein. The fact that these were so similar is to its identity as plasma kallikrein. A role for strong evidence for the identity of plasma kalli- plasma kallikrein in acid activation of inactive krein as the serine proteinase involved in acid renin is therefore apparent. activation. Moreover, plasmin can be excluded The actual way in which the serine proteinase on other grounds, since acid activation is normal works is currently being re-evaluated. The fact in plasma stripped of plasminogen (9, 45) and no that serine proteinases can activate inactive plasmin activity could be detected in our samples renin at pH 7.4 (5, 8-10, 14, 15, 19, 30, 33-39, at 4°C using the selective chromogenic substrate 43, 45, 50) and that serine proteinase inhibitors S2251 in an assay procedure described previously apparently reduced acid activation by ~ 70% (1, (19). It is also noteworthy that plasmas defi- 2, 11, 20, 21) suggested originally that acidifica- cient in factors VII, VIII, X and XI exhibit tion increased serine proteinase activity (by normal acid activation (44). Hill plots of our activating plasma precursors and destroying the data indicate a one-to-one molecular interaction proteinase inhibitor proteins) and that this then of Pro-Phe-ArgCH2Cl. A similar result has activated inactive renin directly after the pH was been reported with the less specific serine pro- readjusted to 7.4 (1, 2). However, this view has teinase inhibitor, aprotinin (Tra.sylol®) (1 1). The been challenged recently by two groups. Hseuh PLASMA KALLIKREIN AND INACTIVE RENIN 557 and Carlson (13) and Leckie and McGhee (23) plasma kallikrein in the activation of inactive have provided evidence that full activation renin in plasma in viva is still unsettled. Hseuh occurs at pH 3.0-3.3 but that this is reversible at et al. (14) consider that prior acidification is alkaline pH and warm temperatures (such as needed to alter the structure of inactive renin occur during the renin assay). Moreover, in the before it can be ‘activated’ by urinary (glandular) presence of serine proteinase inhibitors (apro- and plasma kallikrein, which would obviously tinin (13) and soybean trypsin inhibitor (23)) preclude a role of kallikreins in vivo. Prior they reported that acid activation was followed acidification was also reported to be necessary in by a fall in renin activity during the pH 7.4 step studies of the activation of semi-purified inactive (the same step that was examined in the present renin by pure plasma kallikrein and glandular study using Pro-Phe-ArgCH2Cl). On the basis kallikreins of urinary or pancreatic origin (50). of these results it was proposed that the serine However, Derkx et al. have demonstrated that proteinase (plasma kallikrein) might act either by prior acidification of plasma is not necessary for stabilizing the acid-activated renin (13) or by activation by glandular kallikrein (10, 15). Nor destroying an endogenous renin inhibitor pro- is it needed for activation in amniotic fluid by tein dissociated at low pH (23) or by binding to glandular kallikrein (33) or plasma kallikrein such an inhibitor in competition with renin (23). (present results) and for activation in amniotic In this respect it is of interest to note that long fluid (19, 30, 34-38) or plasma (5, 39) by other chain unsaturated free fatty acids have been serine proteinases. Furthermore, incubation of proposed as having a role in acid activation (18). previously untreated plasma at pH 8.2 at 30°C Interestingly, full activation at pH 3.3 was dem- has been shown to result in a 60% increase in onstrated in the original studies by Morris and renin within 30 min and this increase could be Lumbers using amniotic fluid (38). Since this inhibited by aprotinin (15). Moreover, the fact could be inhibited by pepstatin but not by serine that activation of inactive renin by plasma kalli- proteinase inhibitors, and activation could be krein can indeed take place in the human cir- elicited at pH 4.5 by a pH 1.5-resistant factor, a culation is supported by recent findings of a role for pepsin at pH 3.3 was suggested (6, 19, 38). significant negative correlation between plasma Moreover, in considering the various hypoth- (but not urinary) kallikrein and inactive renin in eses for the mechanism of activation of inactive the plasma of normal human volunteers in dif- renin in plasma it should be noted that the form ferent states of sodium balance (42) and also of of renin activated by acid or proteinases is com- an abnormally high ratio of inactive: active pletely inactive, has a M, of ~ 56,000 (which is renin and impaired acid activation in prekalli- bigger than active renin), and does not decrease krein-deficient plasma (7, 44). The operation of in M, with activation (3, 48, 50). These proper- a plasma-kallikrein-dependent activation mecha- ties are hard to reconcile with the mechanisms nism for inactive renin in the human circulation proposed in references 13 and 23. Furthermore therefore merits careful attention. it may be noteworthy that in the studies men- tioned above (13, 23) the reaction of renin was Supported by grants from the National Health and not stopped prior to radioimmunoassay of angio- Medical Research Council of Australia, the Child- tensin I. Therefore during the equilibration ren’s Assistance Fund and the Australian Kidney Foundation. We thank Rita de Zwart for technical reaction of angiotensin I with antibody (~ 16 hr assistance. at 4°C) more angiotensin I would be generated and other reactions, possibly involving renin, Receivedfor publication 27 Jane 1981 would be taking place as well. This introduces an unnecessary complication into the interpreta- tion of such data. In our assay the reaction is REFERENCES stopped before radioimmunoassay (by boiling) 1. ATLAS S. A., LARAGH J. H. and SEALEY J. E. and we have been unable to reproduce these (1978) Activation of inactive plasma renin: evi- findings. The view that inactive renin represents dence that both cryoactivation and acid-activa- spillover of a biosynthetic precursor, ‘prorenin’, tion work by liberating a neutral serine protease into the circulation has no experimental sup- from endogenous inhibitors. Clfn. Sci. Mol. Med. 55, 135s-138s port, as yet, even though direct evidence has 2. ATLAS S. A., SEALEY J. E. and LARAGH J. H. recently been produced for the biosynthesis of a (1978) “_Acid”- and “cryo”-activated inactive prorenin and its conversion intracellularly to plasma renin. Similarity of their changes during renin (4, 31, 32). B-blockade. Evidence that neutral protease(s) The physiological significance of a role of participate in both activation procedures. Circ. B. J. MORRIS and J. G. McGIRR

Res. 43, Suppl. I, 128-133 methyl ketones. Arclt. Btocltem. Bioplrys. 202, ATLAs S. A., SEALEY J. E., LARAGH J. H. and 420-430 WAREKOIS T. E. (1979) Apparent molecular KETTNER C. and SHAW E. (1978) Synthesis of weight of inactive renin in human plasma. Clin. peptides of arginine chloromethyl ketone. Selec- Res. 27, 466A (abstract) tive inactivation of human plasma kallikrein. CATANZARO D. F. and Moruus B. J. (1980) Cell- Bt'0cliemt'stry 17, 4778-4784 free biosynthesis of renin precursor and activa- KoTcnEN T. A., Wsrcn W. J. and TALWALKER tion by kallikrein and trypsin. IRCS Med. Sci. R. T. (1979) Modification of the enzymatic ac- 8, 495-496 tivity of renin by acidification of plasma and by COOPER R. M., MURRAY G. E. and OSMOND exposure of plasma to cold temperatures. D. H. (1977) Trypsin-induced activation of renin H3-y)et'tenst'on 1, 190-196 precursor in plasma of normal and anephric man. LAWRENCE C. H. and MORRIS B. J. (1981) Mech- Ctrc. Res. 40, Suppl. I, I-171-I-179 anism of activation of inactive renin in human DAY R. P. and MORRIS B. J. (1979) Properties of plasma by puff adder venom. B1'0clu'm. Btoplrys. inactive renin in human plasma. Clfn. Exp. Acta 657, 13-25 Plzarmacol. Plzysfol. 6, 611-624 LECKIE B. J. (1978) An endogenous protease DERKX F. H. M., Boom. B. N., SCI-IALEKAMP activating plasma inactive renin. Clfn. Sci. M. P. A. and SCHALEKAMP M. A. D. H. (1979) Mol. Med. 55, 133s-134s An intrinsic factor XII-prekallikrein-dependent LECKIE B. (1978) Endogenous activator of pathway activates the human plasma renin-angio- plasma-inactive-renin. Lancet ii, 217-218 tensin system. Nature 280, 315-316 LECKIE B. J., MCCONNELL J., MoRToN J. J., DERKX F. H. M., BOUMA B. N., TAN-TJ1oNo H. TREE M. and BROWN J. J. (1977). An inactive L., MAN IN’T VELD A. J., DE BRUYN J. H. B., renin in human plasma. Ctrc. Res. 40, Suppl. I, WENTING G. J. and SCHALEKAMP M. A. D. H. I-4&1-51 (1980) Role of plasma kallikrein in the proteoly- LECKIE B. J. and MCGHEE N. K. (1980) Revers- tic activation of the renin-angiotensin system. ible activation-inactivation of renin in human Clfn. Exp. Hypertension 2, 575-592 plasma. Nature 288, 702-705 DERKX F. H. M., BOUMA B. N., TAN—TJIONG H. LOFTFIELD R. B. and EIGNER E. A. (1969) Mole- L. and SCI-IALEKAMP M. A. D. H. (1979) Activa- cular order of participation of inhibitors (or tors of inactive renin (‘prorenin’) in human activators) in biological systems. Science 164, plasma: their connection with kinin formation, 305-307 coagulation and fibrinolysis. Clfn. Sci. 57, LUMBERS E. R. (1971) Activation of renin in 89s—92s human amniotic fluid by low pH. Enzymologia DERKX F. H. M., TAN—TJIONG H. L., MAN IN’T 40,329-336 VELD A. J., SCHALEKAMP M. P. A. and SCHALE- MATTLER L. E. and BANG N. U. (1977) Serine KAMP M. A. D. H. (1979) Activation of inactive protease specificity for peptide chromogenic sub- plasma renin by tissue kallikreins. J. Clin. strates. Tltrombo. Haenzost. 38, 776-792 Endocrfnol. Metab. 49, 765-769 MCGIRR J. G. and MORRIS B. J. (1980) Effect of DERKX F. H. M., TAN-TJ1oNo H. L. and SCHA]_E'“ selective inactivation of plasma kallikrein by KAMP M. A. D. H. (1978) Endogenous activator Pro-Phe-ArgCH2Cl on the ‘alkaline phase’ of of plasma-inactive-renin. Lancet ii, 218-219 acid activation of inactive renin in human HARA A., MATSUNAGA M., YAMAMOTO J., IVIORI- plasma. IRCS flied. Sci. 8, 683-684 MOTO K., NAGAI H., KANATSU K., PAK C. H., Mortars B. J. (1978) Activation of human inac- Oo1No K. and KAWAI C. (1978) Cryoactivation tive (“pro-”) renin by cathepsin D and pepsin. of plasma renin. Cltn. Sci‘. Mol. Med. 55, 139s- J. Cltn. Endocrinol. Metal). 46, 153-157 141s IVIORRIS B. J . (1978) Properties of the activation HSEUH W. A. and CARLSON E. J. (1981) Revers- by pepsin of inactive renin in human amniotic ible activation of plasma inactive renin. In fluid. Btocltfnt. Btopltys. 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