Action of a human plasma fraction on tetradecapeptide, angiotensin I and angiotensin II

Life Scieaces Vol . 20, pp . 1213-1226, 1977 . Printed in the II .S .A .

Pergama Prees

ACTION OF A HUMAN PLASMA FRACTION ON TETRADECAPEPTIDE, ANGIOTENSIN I AND ANGIOTENSIN II* R. E . Druilhet, M. Overturf, and W. M. Kirkendall Department of Internal Medicine University of Texas Medical School at Houston Houston, Texas 77030 (Received in final form February 24, 1977) A protein fraction designated PF70 was isolated from human plasma and partially purified on Sephadex G-100. PF70 proteins, molecu1ar weight 37,000 to 41,500, formed angiotensin I (AI) and angiotensin II (AII) from 14 C-tetradecapeptide renin substrate (TDP) at 37 C. Hydrolysis was maximal at pH 6.9 but there was no change in the relative quantity of AI and All forn~ed at different pH values . Data indicate that AI was formed first and at a faster rate than AII, but typical converting enzyme activit~r was not detected . Radiolabeled AII was converted to Oes-Asp -angiotensin II (angiotensin III) ; [ 3H]AI was degraded to a single tritiated product, possibly the nonapeptide. These aspartyl hydrolose reactions were apparently inhibited by TDP and were not involved in AI or All generation from TDP . It is concluded that these enzymic activities represent two or more enzymes that are associated with the renin-angiotensin system . The renin-angiotensin systan (RAS) has been thought to be a relativel simple enzymic sequence . The discoveries of active (1-5) and inactive (6-14~ high molecular weight revins, extrarenal renins (19), atypical converting enzymes (15-18), and angiotensin III (20-22) have complicated the concept of the angiotensin II pathway. Recent knowledge of the RAS was gained mainly from animal studies, plasma renin activity (P )measurements, and partially purified enzymes of cadaveric tissue . Relatively few workers have attenpted to extract renin fr~a human plasma (8, 23-25) . The purpose of the present study was to survey haven plasma fractions for renin activity . Materials. Unless indicated, all chemicals used were reagent grade and obtained ~canmercial sources . The 5-L-isoleucine, 3-L-[U 14 C]valine tetradecapeptide renin substrate (TDP) with a specific activity of 4QnC1/mmole was obtained from Schwarz Bio-Research, Orangeburg, New York . Coluim chromatographic materials were obtained from Pharmacia Fine Chemicat~,Piscatoway, N . J . Radiolabeled angiotensin I (5-L-lieu, 10-L-[N3H] 4,5-Le~ ; s . act . 250 mC1/~nnble) and angiotensin II (5-L-[U14 C]Ile; sß act.236 mCi/mmole~ were obtained fr~am New England Nuclear Boston, MA . Angiotensin II homologs, Des-Aspl-, Des-Aspl-Arg 2 -, and Des-Asps-Arg 2-Va1 3 -angiotensin II, were obtained from Schwarz/Mann together with angiotensin I and angiotensin II . Histidylleucine and L-histidine were obtained from Sigma (St. Louis, MO .) . Hunan rnin Standard (MRC, 68/356) was obtained from The World Health Organization, Hempstead, London . *

Supported by National Institutes of Health Grant No . HL-15171 from the National Heart and Lung Institute.

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Plasma Pm tein Action an TDP, AI and All

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Methods Blood: Ten units of human blood were collected in NIH solution A and the p~Îasma was obtained by centrifugation at 1200 q (4 C) for 20 min . Plasma Extraction : A method modified from Overturf et al . (3) was used to extract p asma proteins . All steps were carried out at5~. The plasma (1800 m1) was acidified to pH 2 .5 with 5 N H2S04 and made 0.8 M with respect to NaCI . The mixture was stirred for 30 min and centrifuged at 5000 q for 30 min . The precipitate was discarded and solid (NH4 ) ZSO,, was added to the supernatant to make it 2.5 M. The mixture ~s stirred for 30 min and centrifuged as before . The supernatant was discarded; the precipitate was diluted to 1 .0 M (NHy)2S04 with distilled water, stirred for 30 min and centrifuged at 3000g for 1 h . The supernatant was dialyzed for 24 h against three changes of distilled water, removed from dialysis bags, and centrifuged at 3,000 q for 20 min to remove insoluble protein . Dlsodiun EDTA was added to a final concentration of 0.1 M . The solution was stirred for 5 min and the pH adjusted to 9.5 with 5 N NaOH . The solution was stirred for 30 min and the crude protein fraction was precipitated over 1 hr by the addition of (NHa)2SOa (2 .5 M) . The precipitate was collected by centrifugation at 8,000~ for 1 hr and dissolved in 350 ml cold distilled water . The solution was dialyzed for 36 h against three changes of 0 .005 M phosphate buffer at pH 7.0 . The preparation was again adjusted to pH 2 .5 with 5 N H2Sß, ; each step described above was repeated . The product from the second extraction was mixed with 10 g acid washed aluainum silicate (Kaolin), stirred for 15 min, and centrifuged at 3,000 q for 20 min. The sediment was washed with 30 ml distilled water. The water was discarded. Proteins were eluted from the Kaolin with three 10 ml volumes of citrate-phosphate buffer (0 .002 M, pH 5 .9) . The combined extracts were dialyzed against four changes of 0.005 M phosphate buffer (pH 7.0) and lyophilized . The protein was dissolved in cold distilled water, 20 mg (protein)/ml, and stored at -85 C. Column chromato_--g_raphy : The protein extract was further purified at 5 C by ascen ng ge tration chromatography on Sephadex G-100 columns . The columns (Pharmacia K16/100 and K25/100) were standardized with Blue Dextran 2000 and protein molecular weight standards described earlier (3) . The average partition coefficient (Kay) for each standard was calculated (26) and the values were plotted against the logarithm of the corresponding molecular weight . A regression line ~s constructed by the method of least squares and used to estimate the molecular weight of plasma proteins . A sample (20 mg protein) was applied to the column (1 .6 x 94 an) and proteins ware eluted with 0 .05 M sodium phosphate-0.1 M NaC1 buffer, pH 6 .9 . The buffer flow was maintained at 15 ml/h wfth an LKB 1200 pump . The A2eo of the effluent was monitored (LKB Uvicord II) and 3 ml fractions were collected. The plroteln concentration was determined using crystalline human albumin as standard (27) . Iri each experia~ent, 360 ml of colurtn effluent was collected and the fractions stored (-85 C) until six column runs were made . Tubes in the elution range of 35,000 to 45,000 mol wi; were combined, dialyzed against three changes of distilled water, and lyophilized . The protein wns dissolved in 3 ml phosphate buffer and samples were applied to a second Sephadex G-100 column (2 .5 x 98 cm) . Fractions 70 through 79 (Mol wt = 37,000 to 41,500) were pooled, dialyzed for 24 h against three changes of distilled water and lyophilized . This plasma fraction, PF70, was dissolved in 0.002 M sodium phosphate buffer (pH 6 .9) . The protein content was 70 ug per 10 ul buffer . Bacitracin (0 .001X, w/v) was added to retard bacterial contamination and the fraction was stored at -85 C . AI radiolmaunoass prec p ton met

: Renin substrate was prepared from human plasma by a salt d (28) and further purified on Sephadex 6-100 . The 61,000

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Plasma Protein Action on TDP, AI aad All

1215

to 62,000 molecular wei ht .fraction was dissolved in phosphate buffer (0.002 M, pH 7) and 0 .4 ml, 8 mg ~protain)/m1, wns mixed with 0.1 ml of dialyzed test sample . Four replicates were prepared for each sample . Two of these sarAples were placed at 4 C for 3 h and used to establish levels of endogenous AI . The other samples were incubated for 3 h at 37 C to detect renin activity . Controls consisted of replicate tubes with: 0.4 m1 substrate and 0 .1 ml buffer ; 0.4 ml substrate, 0.1 ml buffer and 175 uGU Human Renin standard; and 0.4 ml buffer with 0.1 m1 test sample. The Squibb Angiotensin I Immutope® kit was used for the assay (29) . Exogenous substrate wns not used for the initial renin assay of pooled plasma . Radiochemical incubations : Plasma fractions were screened for renin activity w t TD . u samp e was incubated at 37 C for 30 or 60 min with 10 ul TDP (550 pmolas) and 10 ul 0.002 M sodium phosphate buffer, pH 6 .9 . The incubations were terminated by freezing at -85 C. The effect of incubation time on the hydrolysis of TDP, [ 3 H]angiotensin I (400 pmoles), and [ 14 C]angiotensin II (40a pmolas) was studied with replicate 30 ul incubation mixtures . The effect of inhibitors was also studied in the system described above . Inhibitors were prepared in 0.002 M phosphate buffer (pH 6.9) and 10 ul was used to replace the incubation buffer. Incubations with substrate, PF70 and buffer alone were used as controls . Tha relationship of pH and the action of PF70 on TDP was determined in 0.002 M NaH2P04/Na2HP04 " 7H20 buffer with pH values from 5 to 8 . Se oration of a tides : The incubation products were separated on Whatmann 3 MM paper 46 x 7 an y 1gh-voltage electrophoresis 1n pyridine-acetic acid buffer, pH 3 .5, for 90 min at 12 C and 2,500 volts (3) . Ninhydrin was used to locate the position of standard peptides and amino acids . Co-electrophoresis of the incubation mixture with [~H]angiotensin I or [ 14C]angiotensin II spotted with, but not incubated with, PF70 was used to confirm the position of hydrolysis products . The mobility of angiotensin II homologs was determined in this manner . Each peptide standard was dissolved in the incubation buffer (2 mg/ml) and 5 ul to 30 ul were elactrophoresed . Substrate radiopurity and possible incubation artifacts were determined by the incubation of substrate, buffer, and inhibitor mixtures . These solutions were electrophoresid and assayed for radioactivity . Radioactivi determination : Radioactive products and the substrate were ocate on t a e ectrop oretogram with a radlochrromatogram scanner (Packard Instrueants, Model 7201) and assayed by liquid scintillation methods . The counting fluid consisted of 2,5-diphenyloxazole (7g), 1,4-bis[2(4-methyl-5phenyloxazole)]benzene (0.7g), and toluene to make 1000 ml . After drying, the 3 MM paper was cut into 5 x 35 an strips, scanned for radioactivity, and the peak areas ware subdivided into 1 x 5 an pieces . Each piece was placed in a vial with 15 ml fluid and counted . Counts were expressed as absolute activity, dpm, (30) . Estimate of PF70 activit : PF70 activity was expressed as a percentage of the su s ra a ra oat v y tatted in individual products . The initial substrate radioactivity and the anwunt recovered after the substrate was electrophoresid were caaparad with experimental values and used to estimate the limitations of the assay . The thanga in product radioactivity relative to controls served as a measure of the inhibitor effect . Inhibition was expressed as a percentage of the amount of l 4 C product foneed in the control incubation less the amount formed with inhibitor, divided by the control level . Results Renin activity was estleated by AI radioiamunoassay at different stages of the extraction sequence. The pooled plasma contained 66 mg protein per ml and

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Plasma Protein Action oa TDP, AI and All

Vol . 20, No . 7, 1977

1 ml formed 12 .8 ng AI in 3 h at 37 C . The specific activity was 0 .19 ng AI/mg protein . Nearly a three-fold increase in renin activity (0 .51 ng AI/mg protein) was obtained with the first extraction ; the protein content was reduced from 119 g to 10 g. The product from the second extraction fornied 0.26 ng AI/mg protein and the kaolin treated product formed 0 .34 ng AI/mg protein . The total protein was reduced to 4.8 g and 0 .93 g by these steps, respectively . The influence of non-renin peptidases on renin recovery was examined using [i 4 C]TDP. Each crude protein sample fornied [ 14C] angiotensin I, but a large number of radiolabeled fragments were detected . The incubations were repeated with 2,3-dimercaptopropanol, 8-hydroxyquinoline and disodium EDTA, but these agents had no significant .effects . A survey of proteins eluted from Sephadex G-100 indicated that most of the activity was present in fractions of molecular weight 35,000 to 45,000 . These proteins formed two radiolabeled products from TDP . The same products were generated after further purification on a second Sephadex G-100 column (Fig . 1) . The greatest activity was present from molecular weight 41,500 (tube 70) to 37,000 (tube 79), PF70 . Co-electrophoresis with peptide standards was used to identify the labeled peptides as angiotensin II (16 cm) and angiotensin I (20 cm) . The mobility of standards on high-voltage paper electrophoresis was expressed relative to L-histidine (Rm) . These values are presented in Table 1 .

cr

FIG . 1 Hydrolysis of 3[U 14C]Val-tetradecapeptide by PF70 at 37 C for 1 hr, pH 6.9 . Radiochromatogram scan of medium after electrophoresis . Replicate mixtures of PF70 (70 ug protein) and [ 1 `'C]TDP (500 pmoles) were incubated for 1 h at 37C and the amount of radioactivity recovered was used to establish the validity of the assay . Controls were also included in the electrophoresis step to verify Rm values . The 14C counting efficiency was 89 t 2 .6~ . On electrophoresis and radiochemical assay, TDP formed a homogeneous peak near the origin with an absolute activity of 9,300 t 276 dpm (n=5) . The total activity recovered from the product and residual substrate peaks, after incubation, was 7,825 t 197 dpm (n=8) ; 1,030 t 260 dpm or 79 t 20 dpm per cm was recovered between peak areas on the electrophoretogram . Non-labeled fragments of TDP, presumably Leull-Va1 12 -Try 13 =Ser 14 and His9-Leuio, could not be detected by this method . However, more than 95X of the initial [ 14 C]valine label was recovered from each incubation mixture. These data and the radiochromatogram profile of incubation media suggest that PF70 contained renin and

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Plasma Protein Action on TDP, AI and All

1217

converting enrsyme activity . TABLE 1 Mobility of peptide standards on paper high-voltage electrophoresis cm from origin

Standard L-Histidine Histidylleucine Des-Aspl-angiotensin II Des-Asps-Arge-anyiotensin II Des-Aspl-Arge-Val 3-angiotensin II Angiotensin I (3H~ Angiotensin II ( 1 C) Tetradecapaptide (3[ 14C]Yal)

32 .4 25 .2 23 .4 22 .5 21 .0 19 .6 16 .3 2 .0

Rm 1 .0 0 .78 0 .72 0 .69 0.65 0.60 0.50 0.06

Results of experiments designed to measure the effect of pH on the PF70 reaction with TDP are shown in Figure 2. Hydrolytic activity was maximal at pH 6 .9, but there was no significant change in the relative amount of AI and All generated at different pH values . wo

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Effect of pH on the PF70 - tetradecapeptide reaction . Fifty mnoles of pepstatin and cupric sulfate and 5 ~anoles of 8-hydroxyquinoline effectively inhibited~TDP hydrolysis (Table 2) but dimercaptopropanol (20 males) and disodium EDTA (4 and 8 Wnoles) were not inhibitory . Sodium chloride (10-3M) had no effect on PF70 activity . In the absence of inhibitors, 16% of the substrate radioactivity was found in the All product and 42% in the AI product . The mobility of standard peptides did not change 1n the presence of inhibitors .

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Plasma Protein Action on TDP, AI and All

Vol . 20, No . 7, 1977

TABLE 2 Inhibition of products formed from tetradecapeptide . Per cent inhibition Angiotensin II

Agent Pepstatin Cupric sulfate Diaodium EDTA 8-Hydroxyquinoline 2,3-Dimercaptopropanol

90 50 0 70 0

Angiotensin I 88 48 0 65 0

The progress curve of TDP hydrolysis (Fig . 3) shows the relative rate of forniatlon of AI and AII . Thasa results were obtained from replicate incubations, each stopped at a predetermined time . Approximately 40~ of the sub strate was converted to AI after 30 min and no significant change in AI was detected thereafter . Tha amount of All forn~ed increased from 16S of the initial TDP concentration at 30 min to 25x at 2 h .

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FIG . 3 Time course of tetradecapeptide hydrolysis . Conversion to angiotensin I (0) and angiotensin II ( ") by PF70 during a two hour period at 37 C, pH 6 .9 . The recovery of tritiated His 9-Leul° from incubations of PF70 with 10[3 H(N)]Leu-angiotensin I (400 pmoles) was used to estimate converting enzyme activity . Tha product fornied from AI (fàn = 0 .82) was not His-Leu (Fig . 4) . Another peptide, different from the AI hydrolysis product, was found in the medium after PF70 and 5[l4C]Ileu-angiotensin II (400 pmoles) were incubated (Fig . 5) . Tha All fragment was electrophoretically identified as the heptapeptide, Des-Asps-angiotensin II (fàn ~ 0.72) .

yol . 2Q, No . 7, 19J7

$lgeaa Protnia l~ct~,on oa TD~~ A~ aad AIx

1219

FIG. 4 Electrophoretic profile of 10[ 3H ]Leu -angiotensin L and PF70 incubated 1 h at 37 C, pH 6.9 .

FIG. 5 Electrophoretlc profile of 5[U14 C]Ileu-angiotensin II and PF~O incubated 1 h at 37 C, pH 6.9 . The amlnop~eptidasa activity of PF70 was studied in a series of timed incubations with [ 4 C]AII, and a second tape curve was obtained wlth [ 3H]AI (Fig . 6) . AI was hydrolyzed faster than All and the relative mobllity of each product remained constant over the 2 h incubation . The substrates were not A 56x counting efficiency was obtained with tritiated randaply fragmented . The mean radlochemlcal recovery, based on zero tlma substrate consamples . centration, wes 96X for [ 3 H]AI and 98X for [14 C]AII . Discussion Established characteristics of the human plasma fraction and enzynes of the RAS ara sum~aarizad in Table 3. The estimated molecular wei ht range of PF70 proteins was close to tonln (16), and human kidney (2,3,15 and plasma (8) renin, but considerably siealler than plasma and lung converting enzynes (31),

1220

Plasma Protein Actioa on TDP, AI and All

Vol . 20, No . 7, 1977

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Vol . 20, No . 7, 1977

Plasma Protai>; Action on TDP, AI ând All

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FI6 . 6 Time course of angiotensin I (0) and angiotensin II ( ") hydrolysis by PF70 at 37 C, pH 6.9 . or angiotensinases (32) . AI and All were hydrolyzed by this fraction but evidence of this activity was absent in the presence of TDP . Hydrolytic activity with the angiotensin substrates, the tentatively identified heptapeptide product from AII, and the absence of labeled His-Leu in 10[ 3H]angiotensin I incubation media suggest the following . The hydrolysis of AII was mediated by an N-terminal hydrolase . This activity was similar to the action of aminopeptidase A isolated from rat kidney microsomes (33) and human blood (34,35) . Angiotensin II heptapeptide and hexapeptide fragments have been detected in human plasma (36) and the possible role of the heptapeptide, "angiotensin III", has bean recently reviewed (22) . The product from AI may also be the result of the N-terminal hydrolase activity and could be the nonapeptide, Des-Asplangiotensin I . Blair-West et al . (37) have discussed this possibility . The nature of the nonapeptide inrelation to the structure and F0n values of DesAspl-angiotensin I would place the theoretical electrophoretic mobility of DesAspl-angiotensin I near the value observed for the AI hydrolysis product . The nonapeptide has been postulated as a possible internlediate in the renin-angiotensin system (22) . Typical converting enzyme activity was not associated with PF70. The plasma proteins formed AI and All from TDP but the N-terminal hydrolase was probably not involved in the reaction . Removal of the N-tenainus from TDP would have resulted 1n Des-Aspl-3[ 14C]Val-T.DP . If this interniediate tridecapep~tide ~s formed initially and further hydrolyzed at Phee-His9 and Leulo-Leu 1 , the tridecapeptide would have fragmented into peptides with electrophoretic tterns of Des-Asps-angiotensin homologs . These fragments would contain [14C]wallne and would have been detected on the radiochranlatogram . If AI and AII were formed first and then hydrolyzed at Asps-Arg 2, labeled Desaspartic acid homologs in addition to the angiotensins would also have been detected . Thus, the aminopeptidase activity detected with AI and All substrates wns believed to be inactive in the presence of TDP .

1222

Plasma Proteia Action on TDP, AI aad All

Vol . 20, No . 7, 1977

Although PF70 has some characteristics in common with RAS enzymes, many of the observed activities are atypical (Table 3) . Tonin is an enzyme from rat submax111ary glands which bypasses the renin step and forms All directly from TDP (16) . It also forms All from AI . Cupric sulfate inhibits the tonin-AI reaction but not the tonin-TDP reaction (16) . Pepstatin and cupric sulfate each Inhibit the hydrolysis of TDP by PF70 but the quantities of AI and All formed are decreased in equal proportions . Like renin, tonin, and human lung converting enzyme (18,40), PF70 is not inhibited by EDTA . Angiotensinases (40,41), lung and plasma converting enzyme (42,43), and PF70 are inhibited by 8-hydroxyquinoline but dimercaptopropanol, which also inactivates angiotensinasas and converting enzymes (11,40-43), has no effect on PF70 . Tonin and the TDP-PF70 reactions are not chloride-dependent . There are two proposed pathways by which Des-AspI-AII (AIII) can be formed (Fig . 7) . The scheme diverges after the forniation of AI and the alternate pathways are initiated either by converting enzyme which leads to AII, the conventional renin-angiotensin scheme, or angiotensinase A leading to the nonapeptide intermediate, Des-Asps-AI . AIII is formed from AI by angiotensinase A or from Des-Asps-AI by converting enzyme (43,44) . The conversions mediated by PF70 can be included in these proposed pathways . PF70 forms AI and All directly from TDP, steps I and II, and acts as angiotensinase A in steps III and IV to produce the Des-Aspl-angiotensin homologs . This activity is sufficient evidence to associate PF70 plasma proteins with the RAS . The observations reported pose the question of whether this is a new activity or the sum of established enzyme activities . The inability to correlate PF70 characteristics with those of known RAS enzymes, or extrarenal "iso renin" (tonln) suggest a new activity . However, additional studies are required to determine homegeneity of enzyme action and the slgnificance of this protein fraction in human plasma . References 1.

L . T . SKEGGS, K . E . LENTZ, J . R . KAHN, and H . HOCHSTRASSER, (Supp . II) 20 and 21 :II-91 - II-100 (1967) .

2.

L . T . SKE66S, K . E . LENTZ, J . R . KAHN, M . LEVINE, and F . E . DORER, Hypertension '72, p . 149-160 Springer-Verlag, New York (1972) .

3.

M . OVERTURF, M . LEONARD, and W . M . KIRKENDALL, Biochem . Pharmacol . 23 :671-683 (1974) .

4.

R . E . DRUILHET, W . M . KIRKENDALL, M . OVERTURF, and R . R . DURRETT, Central A tc ions of s1 amid Related nes, p . 483-490, Pergamon Press, New York 1976 .

5.

M . LEVINE, K . E . LENTZ, J . R . KAHN, F . E . DORER, and L . T . SKEGGS, Circ . Res . (Supp . II)38 :II-90 - II-94 (1976) .

~6 .

B . J . MORRIS and E . R . .LUMBERS, (1972) .

Biochim . Bio

Circ . Res .

s . Acta 289 :385-391

7.

S . L . SKINNER, E . J . CRAN, R . GIBSON, R . TAYLOR, W . A . W . WALTERS, and K . J . CATT, Amer . J . Obstet . ecol . 121 :626-630 (1975) .

8.

R . P . DAY, J . A . LUETSCHER, and C . M . GONZALES, J . Clin . Endocrinol . Metab . 40 :1078-1084 (1975) .

Plasua Protein Action on TDP, AI end All

Vol . 20, No . 7, 1977

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Vol . 20, No . 7, 1977

A . DeLEIVA, A . R. CHRISTLIEB, J . C . MELBY, C . A . GRAHAM, R . P . DAY, J . A .. LUETSCHER, and P . G . ZAGER, New En91 . J . Med . 295 :639-643 (1976) .

10 .

F . H . M . DERKX, G . S . WENTING, A . J . MAN IN'T VELD, J . M . G . v . GOOL, R . P . VERHOEVEN, and M . A . D . H . SCHALEKAMP, Lancet 2 :496-498 (1976) .

11 .

B . J . LECKIE and A . McCONNEI,

12 .

G . W . BOYD,

13 .

M . LAURITZEN, J . J . DAMSGAARD, I . RUBIN, and E . LAURITZEN, J . 155 :317-323 (1976) .

14 .

B . J . MORRIS and C . I . JOHNSTON,

15 .

R . BOUCHER, M . SAIDI, and J . GENEST, Springer-Verlag, New York (1972) .

16 .

R . BOUCHER, J : ASSELIN, and J . GENEST, I-203 - I-209 (1974) .

17 .

A . FITZ . D . BOAZ, and S . WYATT, III-30 (1974) .

Circulation 49 and 40 (Supp . III) :

18 .

D . BOAZ, S . WYATT, and A . FITZ, 495 (1975) .

Biochim . B1o

19 .

D . GANTEN, P . SCHELLING, P . VECSEI, and U . GANTEN . 760-772 (1976) .

20 .

B . S . TSAI, M . J . PEACH, M . C . KHOSLA, and F . M . RUMPUS, Chem . 18 :1180-1183 (1975) .

21 .

A . T . CHIU, J . W . RYAN, J . M . STEWART, and F . E . DORER, Biochem . _J . 155 :189-192 (1976) .

22 .

T . L . GOODFRIEND and M . J . PEACH, Circ . Res . (Supp . I)36 and 37 :I-38 I-48 (1975) .

23 .

J . J . BROWN, D . L . RAVIES, A . F . LEVER, J . I . S . ROBERTSON, and M . TREE, Biochem . J . 93 :594-600 (1964) .

24 .

R . DAY and J . A . LUETSCHER, (1974) .

25 .

R . P . DAY J . A . LUETSCHER, and P . G . ZAGER, 674 (1976 .

26 .

T . S . WORK and E . WORK, Laboratory Techniques in Biochemistr ~d Molecular Bioloav , p . 157 John Wiley, New York ~196~Y

27 .

0 . H . LOWRY, N . J . ROSEBROUGH, A . L . FARR, and R . J . RANDALL,

28 .

5 . SEN, R . R . SMEBY, and F . M . RUMPUS, Biochem . 6 :1572-1581

29 .

E . HABER, T . KOERNER, L . B . PAGE, B . KLIMAN, and A . PURI~DE, Endocrinol . Metab . 29 :1349-1355 (1969) .

Circ . Res .

Circ . Res .

36 :513-519 (1975) .

35 :426-438 (1974) .

Endocrinol .

Biochem .

98 :1466-1474 (1976) .

Hypertension '72 p . 512-523 Circ . Res .

(Supp . I)34 and 35 :

s . Res . Camm .

63 :490-

Am . J . Med . 60 : - _

J . Clin . Endocrinol . Metab .

J . Medicinal _

38 :923-926

Am . J . Cardiol . 37 :667-

J . Biol .

(1967) . J . Clin .

Vol . 20, No . 7, 1977

Plaeaa Protein Actioa on TDP, AI and All

1225

30 .

W. B . BAILLIE,

Adv. Tracer Methodol . 1 :89-92 (1962) .

31 .

J . J . LJWZILLO and B . L . FANBURG, Biochirn . Bio (1976) .

32 .

I . NAGATSU, T. NAGATSU, T. YAMAPgTO, G . G . 6LENNER, and J . W. MEHL, s. Acta 198 :255-270 (1970) . Biochlrn . Bio

33 .

G. G. GLENNER, P. J . McMILLAN, and J . E. FOLK, Nature (Lond) 194:867 (1962) .

34 .

P. A. KHAIRALLAH and I . H. PAGE,

35 .

I . NAGATSU, L . GILLESPIE, J . M. GEORGE, J . E . FOLK, and G . G. GLENNER, Blochern . Phar~nacol . 14 :853-861 (1965) .

36 .

P. F. SEMPLE and J . J . MORTON,

37 .

J . R. BLAIR-WEST, J . P. COGHLAN, D. A. DENTON, J . W. FONDER, B. A. SC066INS, and R . D. WRIGHT, J . Clin . Endocrlnol . Metab. 575-578 (1971) .

38 .

M. M. McKOWN, R. J . WORKWIN, and R. I . 6REGERMAN, 7770-7774 (1974) .

39 .

A. FITZ and M. OYERTURF, New York (1972) .

40 .

G . W. BOYD, A. E. FITZ, A. R. ADAMSON, and W. S . PEART, 213-218 (1969) .

41 .

A. FITZ, G. W. BOYD, and W . S . PEART, Circ . Res .

42 .

Y . S . BAKHLE and A . M. REYNARD, (1971) .

43 .

F. E . DORER, J. R . KAHN, K. E . LENTZ, M. LEVINE, and L . T. SKE6GS, Biochem. Pharniacol . 24 :1137-1138 (1975) .

44 .

A . T . CHIU, J . W . RYAN, J . M . STEWART, and F. E. DORER, Blochem . J . 155 :189-192 (1976) .

Biochem . Med.

s . Acta .

439 :125-132

1 :1-8 (1967) .

Clin . Sçi . Molec . Med .

48 :2 (1975) . 32 :

J . Biol . Chem . 249:

Hypertension '72, p. 507-511 Springer-Verlag, Lancet 1 :

28 :246-253 (1971) .

Nature New Bio1 . (Lond .)

229:187-189,