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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.

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.

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.

Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.

When homogenates of rat liver and hepatomas were centrifuged at 78 000 X g, over 90% of liver N-acetylglucosaminyltransferase assayed with beta-galactosidase- and beta-N-acetylhexosaminidase-treated asialofetuin as acceptor was recovered in the particulate fraction, while as much as 24% of hepatoma transferase was in the supernatant fraction. The particulate transferase solubilized by 0.2% sodium deoxycholate emerged from a DEAE-cellulose column at 0.04 M NaCl (transferase A). The supernatant fractions from all the hepatomas tested contained a second N-acetylglucosaminyltransferase eluted from the column at 0.02 M NaCl (transferase B). Transferase B was absent from liver supernatant fraction. The activities of these transferases toward various acceptors and the effect of beta-N-acetylhexosaminidase on their products suggest that both transferases are UDP-N-acetylglucosamine : alpha-mannoside beta-N-acetylglucosaminyltransferase. Although ovalbumin and glycopeptide V, which was isolated from pronase digest of ovalbumin, were good acceptors, transferase A utilized ovalbumin and glycopeptide V with apparent Km values of 0.44 and 0.33 mM, respectively, whereas the corresponding values for transferase B were 4.5 and 0.050 mM.

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.

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.