Kinya Nakazawa

Subcellular distribution and properties of guanylate cyclase in rat cerebellum

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.

Studies on the demethylation, hydroxylation and N-oxidation of imipramine in rat liver.

Abstract Metabolic pathways responsible for the demethylation, N -oxidation and 2-hydroxylation of imipramine were studied in rat liver in vitro . Isotope-trapping experiments have revealed that imipramine- N -oxide is not involved as an intermediate in the formation of desmethylimipramine from imipramine. The enzyme systems for demethylation and 2-hydroxylation are induced by phenobarbital, but that for N -oxidation is not induced. All available evidence indicates that the demethylation, N -oxidation and 2-hydroxylation reactions may be independent of one another.

Enzymatic properties of phospholipid methylation in rabbit platelets

The enzymatic properties of phospholipid methylation in rabbit platelets were examined using S-adenosyl-L-[methyl-3H]methionine as a substrate. pH optimum for the methylation was around 10.5 under Tris-HCl and glycine-NaOH buffer systems. When Tris-HCl buffer was replaced by phosphate buffer, pH optimum shifted to around 8.0 and the methylation was increased approximately threefold, compared with that in the case of Tris-HCl buffer at pH 8.0. The formation of the 3H-methylated phospholipids was increased by addition of exogenous phosphatidyl-N-monomethylethanolamine or phosphatidyl-N,N-dimethylethanolamine, intermediates of the biosynthesis of phosphatidylcholine from phosphatidylethanolamine. However, the increase in product formations by addition of exogenous intermediates was all but equal under Tris-HCl and phosphate buffer systems at pH 8.0. These results suggest that phosphate ion stimulates the first step of the successive methylation to form phosphatidyl-N-monomethylethanolamine from phosphatidylethanolamine. The methylation in platelets was inhibited to 30% of the basal value with Ca2+ (0.2 mM). However, Ca2+ showed different effects on the methylation in various tissues (activation to 150% of the basal value in the adrenal gland and slight inhibition to 82-88% of corresponding basal values in the liver, lung, kidney and brain).

Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Abstract Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn ++ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg ++ or Mn ++ . The apparent Km values of adenylate cyclase for Mn ++ -ATP and Mg ++ -ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn ++ -GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.

Involvement of a macromolecular activating factor in activity of guanylate cyclase partially purified from supernatant of a pig lung extract.

A guanylate cyclase preparation partially purified from supernatant of a pig lung extract was subjected to affinity chromatography on an Agarose-GTP column. The major portion of the cyclase activity was adsorbed on the column and then eluted with 50 mM EDTA and 0.5 M KCl, whereas the fractions non-adsorbed on the column contained a factor which enhanced the cyclase activity. Addition of the activating factor to a cyclase reaction mixture increase the enzyme activity without a time lag, and this enhancement by the factor was dose-dependent. With concomitant presence of cyclase and the factor in the reaction mixture the apparent Km value for GTP-Mn2+ of the enzyme was 56 microM, this value being the same as in absence of the factor, however, here the maximum velocity increased 4-fold. The factor was nondiffusable, heat-labile, partially sensitive to trypsin, and resistant to acid or alkali. As estimated by gel filtration, this factor had an apparent molecular weight of 85 000.

“EXPERIMENTAL STUDIES ON CEREBRO-HEPATIC RELATIONSHIP — EFFECTS OF SEVERAL CONDITIONS ON ENZYME ACTIVITIES IN RAT LIVER AND BRAIN, AND ON ESTROGEN INACTIVATION IN RAT LIVER —”

1. Due to liver injury induced by carbon tetrachloride, the activities of GPT, GOT, arginase, tryptophan pyrrolase and δ4‐‐3‐ketosteroid reductase in rat liver and the ability of rat liver to inactivate estrogen decreased, while the activities of GPT and GOT in the brain were inversely increased. Decrease in the activities of GPT, GOT and arginase in the liver and the ability of the liver to inactivate estrogen were also to be brought on by forced running or immobilization.