Hisako Kikuchi

Characterization of multiple forms of histone phosphatase in rat liver.

By using chromatography on DEAE-cellulose, aminohexyl-Sepharose 4B and Sephadex G-200, rat liver extract was shown to contain at least three fractions, IA, IB and II, of histone phosphatase. Fractions IA and II are probably the same enzymes as the previously described glycogen synthase phosphatase and phosphorylase phosphatase, respectively, but IB exhibits noticeable activities only with phosphohistone as substrate. Approximate molecular weights of 69 000, 300 000 and 160 000 were determined by gel filtration on Sephadex G-200 for IA, IB and II, respectively.

Activities of sialic-synthesizing enzymes in rat liver and rat and mouse tumors

Abstract 1. 1. Enzymes of sialic acid synthesis were measured in crude extracts of rat liver, a variety of rat hepatomas and mouse Ehrlich ascites carcinoma. 2. 2. These tumors were found to have less than 10% of the UDP-N-acetyl-glucosamine 2′-epimerase and N-acetylmannosamine kinase activities found in liver. N-Acetylneuraminic acid 9-phosphate synthetase and CMP-N-acetylneuraminic acid synthetase were present in liver and tumors in comparable amounts. 3. 3. The level of UDP-N-acetylglucosamine 2′-epimerase was extremely low in fetal liver, but rose sharply during late fetal and early postnatal life. A maximum that was slightly higher than adult values was reached 2 weeks after birth. Throughout these periods, glutamine:fructose 6-phosphate amidotransferase activity remained much higher than adult values.

l-Glutamine:d-fructose 6-phosphate amidotransferase in tumors and the liver of tumor-bearing animals

Abstract 1. 1. l -Glutamine: d -fructose 6-phosphate amidotransferase (EC 2.6.1.16) of rat tissues and rat and mouse tumors was studied in crude extracts prepared in the presence of glucose 6-phosphate of after fractionation of the extracts with ammonium sulfate. 2. 2. In rats bearing Yoshida sarcoma, tumor growth was accompanied by substantial increase both in plasma seromucoid and hepatic amidotransferase activity. In rats bearing AH-130 hepatoma, increase in plasma seromucoid was much less marked and their hepatic amidotransferase activity remained within the control range. 3. 3. Of 11 normal rat tissues studied, liver had by far the greatest amidotransferase activity. However, activities greater than that of liver were found in Yoshida sarcoma, AH-130 and mouse Ehrlich ascites carcinoma. The tumor enzyme was considerably more sensitive to UDP- N acetylglucosamine inhibition than the liver enzyme. 4. 4. Boiled extracts from tumors but not from liver were found to contain an inhibitor specific for amidotransferase. The inhibitor is dialyzable, insensitive to preincubation with UDP- N -acetylglucosamine 2′-epimerase and appears to have been derived from glucose 6-phosphate used for the extraction of amidotransferase.

Hypertriacylglycerolemia and adipose tissue lipoprotein lipase activity in the Nagase analbumineric rat

In Nagase analbuminemic rats, serum triacylglycerol levels were significantly elevated. This abnormality was accompanied by decreased adipose tissue fat stores, and both were more marked in female than in male rats. Parametrial adipose tissue lipoprotein lipase activity was determined in normally fed female rats. When expressed per mg protein, the activity in analbuminemic rats was only 35% of that in control rats. The activity in analbuminemic rats, however, could be increased as in control rats by refeeding starved animals with a fat-free and carbohydrate-rich diet, and the peak values recorded were the same with the two groups. Treatment of animals with streptozotocin lowered adipose tissue lipoprotein lipase activity in both groups to similar levels. These results suggest that hypertriacylglycerolemia associated with analbuminemia may be caused, at least in part, by altered hormonal control of adipose tissue lipoprotein lipase activity.

Transformation fo glucosamine to glycogen and lactate by ascites tumor cells

Abstract 1. 1. Ascites tumors, including Yoshida sarcoma, when incubated in vitro , phosphorylate exogenous glucosamine readily and transform a considerable part of the glucosamine 6-phosphate formed into lactate and glycogen. 2. 2. The metabolic fate of glucosamine differs strikingly according to the concentration of glucosamine employed. When Yoshida sarcoma cells are incubated with 0.5 mM glucosamine, the formation of glucosamine 6-phosphate is slow, and the major end-product is glycogen. There is a lag period of about 5 min before the onset of glycogen and lactate formation. The cellular level of adenine nucleotides is little affected. 3. 3. When the initial concentration of glucosamine is 2 mM, glucosamine is phosphorylated rapidly, and a marked fall in the level of ATP (unaccompanied by a rise in ADP) as well as a large accumulation of glucosamine 6-phosphate results. The major end-product is lactate, the formation of which starts without appreciable lag period. These metabolic differences due to difference in glucosamine concentration arise primarily from a low affinity of hexokinase towards glucosamine. 4. 4. Ascites tumors contain phosphoglucosamine isomerase in much greater amounts than does liver. The enzyme appears to determine the rate of glucosamine conversion to glycogen, lactate and CO 2 in tumor cells.

Inhibition of l-glutamine:d-fructose-6-phosphate amino-transferase by methylglyoxal

Abstract 1. 1. The potent inhibitor of l -glutamine: d -fluctose-6-phosphate aminotransferase (ED 2.6.1.16) present in heated ascites tumor extracts was identified as methyl-glyoxal. It arises from glucose 6-phosphate via triose phosphates. The formation of triose phosphates from glucose 6-phosphate is enzymic, but their conversion to methylglyoxal requires a brief heating. 2. 2. The mode of methylglyoxal inhibition of aminotransferase was studied by using partially purified rat liver enzyme. The inhibition occurs readily and the concentration of methylglyoxal necessary for a 50% inhibition is about 10 μM. The inhibition is non-competitive with respect both to glutamine and fructose 6-phosphate; it is relieved by thiols, of which cysteine was found to most effective, 11 other enzymes tested were not affected by methylglyoxal. 3. 3. The implication of these results for the well-known cytotoxic effect of methylglyoxal was discussed.

Stabilization of glucosaminephosphate synthase from rat liver by hexose 6-phosphates: Properties and interconversion of two molecular forms

Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.