Tatsuo Kokubu

Isolation and characterization of a 34000-dalton calmodulin- and F-actin-binding protein from chicken gizzard smooth muscle

We isolated a 34,000-dalton protein from the heat-soluble fraction of avian smooth muscle using the procedures of ammonium sulfate fractionation, cation exchange chromatography and gel filtration. The amount of 34,000-dalton protein in the muscle homogenate was as much as tropomyosin. The 34,000-dalton protein bound to F-actin and F-actin-tropomyosin in a Ca2+-independent manner, but it Ca2+-dependently interacted with calmodulin. We tentatively named the 34,000-dalton protein gizzard p34K.

Vascular smooth muscle calponin. A novel troponin T-like protein.

In a search for additional Ca2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the Mr range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9=1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Mr=34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOHterminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca2+, but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus.

Serum apolipoprotein A-I, A-II and B levels and their discriminative values in relatives of patients with coronary artery disease.

Serum cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C) and apolipoprotein (apo) A-I, A-II and B concentrations were measured in 109 first-degree relatives of patients with angiographically defined coronary artery disease (CAD). Age- and sex-matched healthy factory employees were chosen as a control group. Male relatives of the CAD patients had significantly higher serum triglyceride and apoB levels, and significantly lower serum HDL-C and apoA-I levels than the controls. Female relatives of the CAD patients also showed similar differences in serum HDL-C, apoA-I and apoB levels. Discriminant analysis indicated that apolipoproteins were better discriminators than lipids in both patients with CAD and their relatives. In univariate analysis, the best discriminator was apoB between male relatives and the controls, and apoA-I between female relatives and the controls. The percentage of exact classification achieved using three variables (serum cholesterol, triglyceride and HDL-C) was 74% in male relatives and 70% in female relatives. By adding variables of apoA-I and apoB, the percentage of correctly classified subjects was increased to 82% and 80%, respectively. These results indicate that serum apolipoprotein abnormalities are prevalent in relatives of the CAD patients. These abnormalities may explain the familial aggregation of CAD.

Clinical significance of measurements of serum apolipoprotein A-I, A-II and B in hypertriglyceridemic male patients with and without coronary artery disease

To examine the relationship of hypertriglyceridemia to coronary artery disease (CAD), we measured serum cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C) and apolipoproteins (apo) A-I, A-II and B in 82 male patients with angiographically defined CAD and 140 age-matched healthy controls. The CAD patients had significantly lower apo A-I and A-II and HDL-C levels, but had higher apo B and triglyceride levels than the controls. After adjustments of apolipoproteins for serum triglyceride, CAD patients had significantly higher apo B and lower apo A-I and A-II levels than the controls. Discriminant analysis showed that apo B was the best discriminator and that apo A-I was next. In the normotriglyceridemic subgroup HDL-C also had a sufficient power for discrimination between CAD patients and the controls, but in the hypertriglyceridemic subgroup HDL-C had no discriminative power. Both apo A-I and B had significant discriminative power between CAD patients and the controls, independently of the serum triglyceride level. These results indicate that measurements of serum apo A-I and apo B are useful for the study of coronary risk factor in hypertriglyceridemic subjects. Finally, it is necessary to sub-classify dyslipoproteinemia by serum apolipoprotein levels for predicting the future occurrence of CAD in the general population.

Occurrence of anti-gizzard P34K antibody cross-reactive components in bovine smooth muscles and non-smooth muscle tissues

In our previous paper (Biochem. Biophys. Res. Commun. 141, 20-26 (1986) we reported the isolation of a 34000-dalton protein (p34K) which binds to calmodulin and F-actin from chicken gizzard smooth muscle. We examined the distribution of the immunoreactive component of gizzard p34K in bovine tissues by immunoblot analysis using a rabbit polyclonal antibody raised against gizzard p34K. The immunoreactive components with molecular weights of 33000-35000 were detected in all smooth muscles from aorta, esophagus, stomach, trachea and uterus. In non-smooth muscle tissues, a 36000-dalton cross-reactive protein was present in adrenal medulla and cortex. The immunoreactive form of gizzard p34K occurred in large amounts in smooth muscles from various bovine tissues.

Effects of captopril (SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats

High doses of captopril (SQ 14, 225) (120-160 mg/kg/day) were administered orally to normal rats, and the effects on the renin-angiotensin-aldosterone system were observed. Plasma angiotensin converting enzyme (ACE) activity was elevated significantly on the 3rd, 7th and 30th days of captopril administration. ACE activity in the lung and the kidney was significantly decreased on the 1st day then gradually increased, becoming significantly higher than that of controls by the 30th day. Plasma renin activity (PRA) was significantly elevated on the 1st day and remained at a high level until the 30th day. Renal renin content was found to be significantly lower on the 1st and 3rd days. Plasma aldosterone concentration was not affected by captopril treatment, whereas serum potassium concentration was found to be significantly lower on the 1st, 3rd and 30th days. It is suggested that besides its inhibitory action on ACE, captopril has a direct or indirect stimulating action on ACE production as well as on renin release.

A novel troponin T-like protein (calponin) in vascular smooth muscle: interaction with tropomyosin paracrystals.

Calponin is a smooth muscle tropomyosin-binding protein which is antigenically related to troponin T. The fine localization of calponin and troponin T on the polar paracrystals of smooth muscle tropomyosin were studied by electron microscopy. Both calponin and troponin T bound to the filamentous tropomyosin at a site about 17 nm away from the C-terminal end of tropomyosin molecule. This calponin-binding site corresponds to the domain which is highly conserved in the amino-acid sequences among the tropomyosin isoforms from skeletal muscle and smooth muscle. The result suggests that calponin, in collaboration with tropomyosin, may play an important role in regulating contractile apparatus.

Peptide Inhibitors of the Renin–Angiotensin System

RENIN is said to split a leucyl–leucine bond in alpha 2 globulin substrate—angiotensinogen—to yield a decapeptide, angiotensin I. Angiotensin I is subsequently converted into an octapeptide, angiotensin II, by a converting enzyme contained in plasma. Angiotensin II has a pressor activity. We have studied the effects of synthetic peptides with a leucyl–leucine bond and their derivatives on the activity of renin in the formation of angiotensin. The peptides were di-, tri-, tetra- and pentapeptides with leucyl–leucine bonds at the C-terminal end, in the middle position, at the N-terminal end or with one of the other structures as shown in Table 1.

Imidapril hydrochloride in essential hypertension : a double-blind comparative study using enalapril maleate as a control

Objective: To assess the value of using imidapril hydrochloride (ACE/TA-6366), a long-acting angiotensin converting enzyme (ACE) inhibitor developed in Japan, to treat patients with essential hypertension. Patients and methods: A double-blind, comparative, phase III study was carried out using enalapril maleate as a control, with a 4-week observation period and a 12-week treatment period. Both drugs were started at a dose of 5 mg once a day, increasing to 10mg in patients whose antihypertensive response was insufficient after 4 weeks. The study included 231 outpatients aged 30-74 years; of these, 108 in the imidapril group and 115 in the enalapril group were assessed. There were no differences in background factors between groups. Results: An adequate antihypertensive effect was observed in 71.3% (77/108) in the imidapril group and in 66.1% (76/115) in the enalapril group, with no significant difference between groups. The pulse rate was unchanged in both groups. The drug had no adverse effects in 86.1% (93/108) of the imidapril group and 79.1% (91/115) of the enalapril group, with no significant difference between groups. Adverse drug effects were observed in 5.6% (6/108) of the imidapril group and 12.2% (14/115) of the enalapril group. Cough was the most frequent side effect, reported in 0.9% (1/108) of the imidapril group and 7.0% (8/115) of the enalapril group. Other side effects were reported in 4.6% (5/108) of the imidapril group and 5.2% (6/115) of the enalapril group. Abnormal laboratory values were observed in 3.7% (4/108) of the imidapril group and 0.9% (1/115) of the enalapril group. Conclusions: Imidapril showed excellent clinical efficacy and safety compared to enalapril. The low incidence of cough is of particular interest.

Angiotensin I-converting enzyme in human urine

It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.