Shigeru Tsuiki

Glycogen synthase phosphatase of rat liver. Its separation from phosphorylase phosphatase on DE-52 columns

Abstract Glycogen synthase D was prepared from rat liver by chromatographing the glycogen pellet on DE-52 columns. It was free of glycogen and phosphorylase and converted readily into synthase I upon incubation with glycogen synthase phosphatase. With this synthase D as substrate, the identity of rat liver glycogen synthase phosphatase was studied by means of DE-52 column chromatography. Under the conditions developed, synthase phosphatase emerged from the columns as a sharp, single peak, and phosphorylase phosphatase came off later. The two phosphatases were also different from each other in stability, synthase phosphatase being less stable than phosphorylase phosphatase.

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.

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.

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GDla and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo‐GM2 and asialo‐GM1, respectively, by thin‐layer chromatography.

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.

Studies on glycogen synthase D phosphatase of rat liver—multiple nature

Abstract More than 70% of the glycogen synthase D phosphatase activity of rat liver homogenate was recovered in the postmicrosomal supernatant. Using the phosphatase preparations partially purified from the supernatant, conditions have been developed whereby the conversion of rat liver glycogen synthase D into synthase I is proportional both to time and to phosphatase concentration. The partially purified D phosphatase was resolved into six peaks by the use of DEAE-cellulose chromatography. Sephadex G-200 chromatography resolved the same phosphatase into three fractions with molecular weights of approx. 1.15·10 5 , 3.4·10 5 and above 5·10 5 , respectively. The six phosphatase forms separated by DEAE-cellulose chromatography were not identical to each other with respect to molecular weight and tissue distribution. These findings suggest that multi-isozymic forms of synthase D phosphatase exist in rat liver.

Purification and properties of UDP-N-acetylglucosamine 2′-epimerase from rat liver

Abstract 1. 1. UDP -N- acetylglucosamine 2′-epimerase (EC 5.1.3.7) has been purified 500-fold from rat liver with UDP as stabilizing agent. In the presence of UDP and dithiothreitol, the final preparation is stable and loses only 20% of the initial activity over 3 days storage at 4 °C. 2. 2. UDP and UDP -N- acetylglucosamine protect 2′-epimerase from inactivation by aging. UDP also is a competitive inhibitor, the K i being 0.14 mM. Uridine, the stabilizing agent used by previous workers, neither stabilized nor inhibited the enzyme. It is suggested that UDP interacts with the enzyme at the substrate site. Dithiothreitol also stabilizes the enzyme but only under limited conditions. 3. 3. 2′-Epimerase exhibits negative cooperativity in UDP -N- acetylglucosamine binding. The apparent K m value in the lower activity range is 0.08 mM. The negative cooperativity is abolished by UDP or CMP -N- acetylneuraminic acid. 4. 4. 2′-Epimerase is highly sensitive to inhibition by CMP -N- acetylneuraminic acid; the concentration required for 50% inhibition (0.025 mM) is close to its intracellular levels. A Hill plot yielded the Hill coefficient ( n H ) of 5.7. 5. 5. When UDP but not dithiothreitol is present, the purified enzyme undergoes alterations in UDP -N- acetylglucosamine binding such as the disappearance of negative cooperativity, an increase in K m and the appearance of substrate inhibition; all are reversed by dithiothreitol.

Effect of phosphoglucose isomerase and glucose 6-phosphate on UDP-N-acetylglucosamine inhibition of L-glutamine-D-fructose 6-phosphate aminotransferase.

1. 1. l-Glutamine:d-fructose 6-phosphate aminotransferase (EC 2.6.1.16) was purified about 30-fold from rat liver extracts with a recovery of about 70%. Fractionation involved (NH4)2SO4 precipitation and DEAE-Sephadex column chromatography. Further purification (240-fold) was possible by use of hydroxylapatite chromatography. 2. 2. The sensitivity of aminotransferase to UDP-N-acetylglucosamine inhibition was markedly reduced when the (NH4)2SO4 precipitate was purified by DEAE-Sephadex column chromatography. The original high sensitivity, however, was restored by addition of phosphoglucose isomerase (d-glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) or glucose 6-phosphate. 3. 3. Glucose 6-phosphate enhanced the UDP-N-acetylglucosamine inhibition of aminotransferase purified by DEAE-Sephadex chromatography. The kinetics of UDP-N-acetylglucosamine inhibition with respect to fructose 6-phosphate were of the competitive type in the purified preparation but of the mixed type in the ammonium sulfate precipitate. The former preparation, however, exhibited the mixed-type kinetics when fructose 6-phosphate/glucose 6-phosphate ratio was maintained at 0.32, the equilibrium of phosphoglucose isomerase reaction. 4. 4. It. was concluded that the higher sensitivity to UDP-N-acetylglucosamine observed in crude preparations of aminotransferase is due to phosphoglucose isomerase which converts fructose 6-phosphate added as substrate to glucose 6-phosphate. 5. 5. Comparison of the rat liver and Yoshida sarcoma aminotransferases purified by DEAE-Sephadex chromatography confirmed the previous observation that the tumor enzyme is more sensitive to feedback inhibition than is the liver enzyme.

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.

Glycogen synthesis and glycogen synthetase in rat ascites hepatomas of low and high glycogen content.

Abstract 1. 1. Glycogenic capacities of two rat ascites hepatomas, namely, glycogen- deficient AH-130 and glycogen-rich AH-66F, were compared either at whole cell level with [14C]glucose as substrate or by studying the activity and properties of glycogen synthetase. 2. 2. With [14C]glucose as substrate, glycogen synthesis was initiated in the two hepatomas at comparable rates, but in AH-130 the rate decline as glycogen accumulated while in AH-66F the rate was little affected by cellular glycogen level. 3. 3. When 2 mM amytal was present, AH-130 failed to synthesize glycogen from glucose while AH-66F carried out this synthesis at substantial rates. In AH-66F, lower concentrations of amytal led to the stimulation of glycogen synthesis; similar effects were produced by rotenone or 2,4-dinitrophenol. 4. 4. In glycogen-rich AH-66F cells, about 90% of the glycogen synthetase was recovered in particular fraction in association with glycogen. The enzyme was totally in a glucose 6-phosphate-independent (I) form. These findings suggested that the glycogen-induced I to D (glucose 6-phosphate-dependent) conversion of glycogen synthetase, previously reported to occur in AH-130, might not operate efficiently in AH-66F.