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Biomedical subjects

T Kusaka

Publications and source records attributed to T Kusaka.

At least 73 records · Page 4Linked to original sources
PubMed1981

Two esterases released from Mycobacterium smegmatis from the hydrolysis of long chain acyl-CoAs and Tween.

An esterase activity hydrolyzing palmitoyl-CoA was released into the culture medium from Mycobacterium smegmatis. Although another esterase activity hydrolyzing Tween 20 (polyoxyethylene sorbitan monolaurate) was also found in the culture medium, the bulk of the esterase activity was retained in the cells. However, treatment of early-log phase cells with lysozyme to prepare ghosts released 80% of the Tween 20 hydrolyzing activity, indicating the localization of the esterase in the periplasmic space or the cell envelope fraction. The presence of two different esterases hydrolyzing palmitoyl-CoA and Tween 20, suggested by the above results, was confirmed by the separation of these esterases on phenyl-Sepharose column chromatography. Palmitoyl-CoA hydrolase (thioesterase) was purified 630-fold from lysozyme-treated supernatant fluid to homogeneity, by means of Sephadex G-100 gel filtration, and DEAE-cellulose, phenyl-Sepharose and Blue-Agarose column chromatographies. Its molecular weight was approximately 42,000. Tween hydrolase was partially purified 150-fold by the same purification procedure up to the step of phenyl-Sepharose chromatography and its molecular weight was found to be about 51,000. These activities were stable against heating at 60 degrees C and treatment with non-ionic detergents. Thioesterase hydrolyzed long chain acyl-CoAs (C12-C20), but not Tween 20-80 or beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate, but not acyl-CoAs. Both esterases hydrolyzed monoolein, but not diolein, triolein, or phosphatidylcholine.

Acyl Coenzyme A↗
PubMed1981

Isolation and identification of mycolic acids in Mycobacterium leprae and Mycobacterium lepraemurium.

Mycolic acids with a characteristic structure were isolated by high performance-liquid-chromatography (HPLC) and mass-spectrometry from a foot pad of a nude mouse inoculated with Mycobacterium leprae. Mycolic acids with the same structure were also obtained from mycobacteria collected from the liver of an armadillo with experimental leprosy. Mycolic acids were isolated from Mycobacterium lepraemurium grown both in vivo and in vitro and these mycolic acids had different structures from those of M. leprae. Mycolic acid structures have great taxonomical significance. The methods used for isolating and analyzing mycolic acids appear applicable for the rapid identification of M. leprae in samples containing at least 10(9)-10(10) mycobacterial cells. Using our method, mycolic acids with the same structure were found in mycobacteria from armadillos experimentally infected with M. leprae and from armadillos with naturally acquired leprosy-like disease. It is likely, therefore, that the pathogenic mycobacteria of the naturally acquired disease are the same as, or at least closely related to, M. leprae. The present work suggests that M. leprae has a special position in mycobacterial phylogeny.

Animals↗
PubMed1981

Purification and characterization of 2-enoyl-CoA reductase of Mycobacterium smegmatis.

2-Enoyl-CoA reductase was purified to homogeneity for the first time from the crude extract of Mycobacterium smegmatis. Its molecular weight was estimated to be 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted as an electron donor for the reduction of 2-enoyl-CoA, while NADPH did not. The Km value for NADH was 21.3 microM. On the other hand, NAD inhibited the reaction for competing against NADH, as the Ki value for NAD was 47 microM. Among the enoyl-CoAs used as substrates, those having C10-C16 were found to be most suitable substrates for the purified reductase in terms of both apparent Km and Vmax values. The enzyme was strongly inhibited, however, when the concentration of the C16-substrate was over 50 microM. The enzyme had almost no activity towards substrates having less than C8. When NAD3H was used as an electron donor to 2-dodecenoyl-CoA in the presence of the purified reductase, only laurate was tritiated as the product. Diacetyl and phenylglyoxal, agents that react specifically with arginine, inactivated the reductase in a time- and concentration-dependent manner during the preincubation. These results suggest that some arginine residues in the reductase protein are involved in the enzyme activity.

Diacetyl↗
PubMed1980

Purification of two forms of enoyl-CoA hydratase from Mycobacterium smegmatis.

Two forms of enoyl-CoA hydratase (hydratases I and II), which are different from each other in substrate specificity, were found in a crude extract of Mycobacterium smegmatis. Hydratase I was more active with crotonyl-CoA as a substrate than with decenoyl-CoA, whereas the reverse was the case for hydratase II. Hydratase I was purified 688-fold to homogeneity with a yield of 14.5% from the crude extract. Its molecular weight was estimated to be 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 30,000 by gel filtration, suggesting that the enzyme is dimeric. Hydratase II was also partially purified. The Vmax of hydratase I decreased progressively with increase in the carbon-chain length of the substrate from 2,488 units/mg for crotonyl-CoA to 154 units/mg for hexadecenoyl-CoA, whereas the Km values for crotonyl-CoA (82 microM), decenoyl-CoA (91 microM), and hexadecenoyl-CoA (105 microM) were similar. Both hydratases were inhibited by acetoacetyl-CoA and pCMS, but not by N-ethylmaleimide or monoiodoacetate.

Enoyl-CoA Hydratase↗
PubMed1980

Involvement of one of two enoyl-CoA hydratases and enoyl-CoA reductase in the acetyl-CoA-dependent elongation of medium chain fatty acids by Mycobacterium smegmatis.

2-Enoyl-CoA reductase was purified 150-fold from the crude extract of Mycobacterium smegmatis. The purified reductase required NADH, but not NADPH, as a reductant and catalyzed the reduction of C4 to C16 enoyl-CoAs, though the activities toward shorter chain substrates (c4 and C6) were very low. Thiolase was also partially purified from the same source. These two enzymes were used, together with 3-hydroxyacyl-CoA dehydrogenase and the two forms of enoyl-CoA hydratase (hydratases I and II) previously purified from the same source, to reconstitute fatty acid elongation activity. The products formed from [1-14C]acetyl-CoA and decanoyl-CoA in this reconstituted system were analyzed by thin-layer chromatography and radio-gas-liquid chromatography. The system containing hydratase II produced laurate and 3-hydroxylaurate (in the form of their CoA esters) and the ratio of laurate to 3-hydroxylaurate increased as the incubation time was increased. The system containing hydratase I produced only 3-hydroxylaurate. 3-Hydroxylaurate was also the only product when enoyl-CoA reductase was omitted from the system containing hydratase II. It is concluded that hydratase II, but not hydratase I, is functional in fatty acid elongation by M. smegmatis and that enoyl-CoA reductase is also essential for the reaction. CoA and NAD+ inhibited the reconstituted elongation activity in competition with acetyl-CoA and NADH, respectively.

Acetyl Coenzyme A↗
PubMed1979

Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis.

3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel filtration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereospecificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i.e., 6.0 for the oxidation of NADH and 9--10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i.e., esters of CoA, pantetheine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4 mM, respectively. Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The antibodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1979

Change in plasma renin activity by cold storage of plasma in normal subjects and patients with essential hypertension and primary aldosteronism.

The time courses of change in renin activity after cold storage of human plasma at -5 degrees C and pH 7.4 were examined in 5 normal subjects, 6 patients with essential hypertension and one female patient with primary aldosteronism before and after extirpation of the adrenal tumor. In the 5 normal subjects and 6 essential hypertensives, the gradual increase in plasma renin activity was observed until 10 days of cold storage. The same result was obtained in the case of primary aldosteronism. However, there was no increase in renin activity despite of cold storage for 10 days in plasma which was sampled from this patient 45 days after operation. These data indicate that a period of 4 days for cryoactivation of human plasma renin as has been reported by Sealey et al. is not sufficient to accomplish activation of renin by cold storage.

Adolescent↗
PubMed1978

Effect of furosemide on urinary kallikrein excretion in patients with essential hypertension.

The effect of furosemide on urinary kallikrein excretion was studied in 10 patients with essential hypertension and 9 normal volunteer subjects. After intravenous administration of furosemide and 2 hours of upright posture, urine volume (UV), urinary sodium (UNaV) and potassium (UKV) excretion, plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary kallikrein markedly increased. However, the augmentation of urinary kallikrein in patients with essential hypertension (1.50 +/- 0.19 EU/2 hr) was less remarkable than that in normal subjects (2.33 +/- 0.24 EU/2 hr), although the same degrees of response were observed in PRA and PAC. The increments of UV, UNaV and UKV in patients with essential hypertension were also significantly lower than in normal subjects. Significant positive relations were found between urinary kallikrein and UV or UNaV in both hypertensive and normotensive groups, but there was no such correlation before fursemide administration. It is likely that diuresis and natriuresis induced by furosemide are somehow associated with an increase in urinary kallikrein excretion. Blunted response of urinary kallikrein in essential hypertension may suggest an abnormality in the renal kallikrein-kinin system in this disease.

Adult↗
PubMed1978

Taxonomic study and fermentation of producing organism and antimicrobial activity of mildiomycin.

A taxonomic study of strain B-98891, which produced an antibiotic effective against powdery mildew of barley, identified it as Streptoverticillium rimofaciens. On agar media the antibiotic, which was named mildiomycin, was only weakly active against most fungi and bacteria tested. However, it inhibited some Mycobacterium and Rhodotorula, and it showed excellent control of powdery mildew of barley plants in greenhouse tests at concentrations between 31.2 and 62.5 ppm. Rhodotorula rubra IFO 0907 was selected as the test organism for in vitro assay of mildiomycin.

Antifungal Agents↗
PubMed1977

Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis.

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.

Acetyl-CoA C-Acetyltransferase↗