Kunimi Kikuchi

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.

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.

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.

Identification and characterization of Mg2+-dependent phosphotyrosyl protein phosphatase from rat liver cytosol

Although highly purified preparations of Mg2+-dependent phosphoseryl protein phosphatase (also designated phosphatase IA or phosphatase 2C) dephosphorylated phosphotyrosyl histone, the activity has been resolved from phosphatase IA by polyacrylamide gel electrophoresis at pH 9.5. This novel phosphotyrosyl-specific protein phosphatase absolutely requires Mg2+ or Mn2+ for activity, is inhibited by Zn2+, vanadate and fluoride, and has an optimal pH of 9.0 and Mr = 50,000. Certain properties of this phosphatase so closely resemble those of phosphatase IA that the two enzymes tend to be copurified through various separation procedures.

Metabolism of exogenous N-acetylglucosamine in extracts of rat kidney, liver and hepatoma

1. The metabolism of exogenous N-acetylglucosamine (GlcNAc) in rat kidney extracts was greatly stimulated by fructose 1,6-diphosphate (Fru-1,6-P2) and to a lesser extent by phosphoenolpyruvate. They served as a generator of ATP. Under these conditions, the majority of metabolized GlcNAc was recovered in the form of glycolytic intermediates. 2. The metabolism of exogenous GlcNAc in rat liver extracts was stimulated by phosphoenolpyruvate but not by Fru-1,6P2. With phosphoenolpyruvate present, most of the metabolized GlcNAc was recovered as sialic acid. 3. The metabolism of exogenous GlcNAc in rat hepatoma (AH-130) extracts was stimulated by Fru-1,6-P2 and to a lesser extent by phosphoenolpyruvate. Even with phosphoenolpyruvate present, the synthesis of sialic acid was extremely small. In these respects, hepatoma extracts resemble kidney extracts rather than those of liver.