Kunio Hiwada

Purification and properties of endopeptidase from rabbit red cells and its process of degradation of angiotensin

Abstract Endopeptidase, showing angiotensinase activity in rabbit red cells, was purified by fractionation with (NH4)2SO4, DEAE-cellulose column chromatography and Sephadex G-200 gel filtration. The specific activity of angiotensinase on the purified preparation was increased about 8000-fold compared with that of the crude hemolysate. The peptide bonds cleaved by the enzyme preparation in [α- l -Asp1, Ile5]-angiotensin II were Arg-Val, Tyr-Ile and Ile-His. It was demonstrated that hydrolysis of the Tyr-Ile bond with the enzyme was the first step in the inactivation process of angiotensin. The results indicated that the purified so-called angiotensinase might be an endopeptidase without hydrolytic activity on casein, p- toluenesulfonyl - l - arginine methyl ester and acetyl- l -tyrosine ethyl ester.

Angiotensinase in red cells.

Abstract Comparison of angiotensinase activity in red cells and plasma from rabbits and the fractionation of the enzyme were studied. 1. (1) Angiotensinase activity in red cells from humans and rabbits was much higher than that in plasma. Angiotensinase activity in red cells and plasma, when horizontal starch block electrophoresis was employed, was the highest in the albumin position under the conditions employed. In red cells, two additional fractions showing angiotensinase activity were demonstrated in the position toward the anodal side further than the albumin position. 2. (2) In order to isolate the enzyme in the albumin position in red cells from rabbit, fractionation with ammonium sulfate and DEAE Sephadex A-50 column chromatography were made. Specific activity of angiotensinase in the purified preparation was increased about 300-fold or more compared with that of the crude hemolysate. It was shown that the activity of angiotensinase was not due to leucine aminopeptidase.

Role of glandular kallikrein in the activation process of human plasma inactive renin.

Completely inactive renin was isolated from normal human plasma by DEAE-Sepharose column chromatography and Blue-Sepharose column chromatography. This inactive renin had a molecular weight of 54,000 daltons as determined by gel filtration on Ultrogel AcA 44. When the inactive renin was activated by trypsin, its molecular weight decreased to 48,000 daltons. The trypsin-activated renin differed from a native form of active renin in plasma with respect to molecular weight (active renin, 43,000), pI value (active renin, 5.20; trypsin-activated renin, 5.06), km value (active renin, 60 nmoles/liter; trypsin-activated renin, 89 nmoles/liter), Ki value for pepstatin A (active renin, 2.6 mumoles/liter; trypsin-activated renin 5.0 mumoles/liter) and pH profile for angiotensin formation. Glandular kallikrein (human urinary or pig pancreatic) did not activate the inactive renin. When the trypsin-activated renin was treated with glandular kallikrein, its activity was unchanged, but its molecular and kinetic properties except pI value (trypsin-activated kallikrein-treated renin, 4.82) coincided with those of a native form of active renin in plasma. These results indicate that glandular kallikrein does not directly activate inactive renin but participates in the activation process of inactive renin. The results also suggest that inactive renin in human plasma is a renin precursor.

A Radioimmunoassay for the Measurement of Aminopeptidase (Microsomal) in Human Serum

A radioimmunoassay for the measurement of aminopeptidase (microsomal) (AP) in human serum was developed by using antiserum to human kidney AP. AP purified from kidney and AP present in normal serum and in serum from a patient with obstructive jaundice gave parallel logit-log transformation lines, suggesting immunological identity. The mean concentration of AP in normal serum (n = 104) was 1.33 +/- 0.30 (mean +/- SD) micrograms/ml. Men had significantly higher serum AP levels (1.41 +/- 0.30 micrograms/ml) (p less than 0.005) than women (1.24 +/- 0.28 micrograms/ml). Serum AP levels of patients with hepatoma (2.26 +/- 0.87 micrograms/ml) and cancer of the pancreas or the biliary tract (2.90 +/- 0.67 micrograms/ml) were significantly higher (p less than 0.005) than those of normal subjects. Patients with acute and chronic hepatitis (2.06 +/- 0.66 micrograms/ml) also had significantly higher serum AP levels (p less than 0.005) than normal subjects. In pregnant women, however, the increase in AP activity without the increase in AP concentration showed that the increased AP activity was due to an enzyme other than AP. The enzyme levels and activities in normal serum as well as in patients sera were significantly correlated (normal, r = 0.77; patients, r = 0.95). Based on the specific activity of AP purified from human plasma, the enzyme activity splitting L-alanyl-beta-naphthylamide is due almost completely to AP in normal subjects and in patients with hepatobiliary diseases.

Inhibitory effect of bile acids on renin angiotensinogen reaction system

Die Wirkung verschiedener synthetischer Derivate der Gallensäure wurden auf die Reninaktivität in Form des Angiotensin in vitro untersucht und es wird darauf hingewiesen, dass die 7-Desoxyform der Gallensäuren für die Renininhibition wesentlich ist.

Antihypertensive effect of purified enzyme showing angiotensinase activity from rabbit red cells in rats

In der akuten Phase der Goldblatt-Hypertonie der Ratte verhinderte die Injektion von reiner Angiotensinase ein Aussteigen des Blutdruckes. Die Behandlung war unwirksam bei chronischem Hochdruck.

Plasma angiotensinase activity in liver disease

Abstract The electrophoretic detection of the appearance of plasma angiotensinase activity in the γ-globulin zone, named angiotensinase G, in patients with liver damage was studied using rabbits. Angiotensinase G could not be detected either in liver extract from normal rabbits or from rabbits with experimental liver damage. When normal rabbit plasma was incubated together with liver extract, angiotensinase G activity was demonstrated electrophoretically in the incubation mixture. In vitro experiments indicated that γ- or β- globulin fractions of plasma and a heat unstable substance in microsome fractions of liver cells were essential for the formation of angiotensinase G.

Effects of exogenous renin and sodium load on renin activity in unilaterally nephrectomized rats

SummaryEffects of injection of homologous renin and sodium load given through 1.5% saline as drinking water on plasma renin activity and renin content of the kidneys were studied in rats with intact kidneys (group A) and in unilaterally nephrectomized rats (group B). During an experimental period of 10 days, blood pressure was measured four times. At the end of the experiment, the plasma renin activity and the renal content of renin were determined.Renin injection did not cause a rise in blood pressure in both groups. In unilaterally nephrectomized rats, renin content of the remaining kidney increased significantly. This increase was suppressed either by the renin injection or the salt load, whereas neither the renin injection not the salt load affected renal content of renin in group A. In unilaterally nephrectomized rats, the renin injection or the sodium load induced a significant decrease in plasma renin activity when compared to rats in group A.