Glycogen phosphorylase isoenzymes from hepatoma 3924A and from a non-tumorigenic liver cell line. Comparison with the liver and brain enzymes

Abstract

Glycogen phosphorylase isoenzymes were isolated from normal rat liver, rat brain, the glycogen-poor Morris hepatoma (MH) 3924A, and the glycogen-rich non-tumorigenic liver cell line C1I. Electrophoretic and immunological characterization of the enzymes showed that tumour and C1I cells expressed a phosphorylase isoform similar to the brain type; the liver type was not detectable. All enzymes were obtained as dimers; the Mr of the subunits was 96000 (liver), 93000 (brain and MH 3924A) and 92000 (C1I). Isoelectric focusing revealed a main band of pI 6.34 for liver phosphorylase a, pl 5.67 for the enzymes from MH 3924A and brain, and pI 5.68 for CII phosphorylase. Partial kinetic characterization of the AMP-independent forms of the isoenzymes yielded Km values for glucose 1-phosphate of 3.5 + 0.5 mm (liver), 3.9 mm (brain), 1.9 + 0.3 mm (MH 3924A) and 2.5 + 0.5 mm (C1I); Km values for glycogen were 0.4 mM (liver) and 0.3 mm (MH 3924A and C1I), calculated as glucose equivalents. The AMP-independent phosphorylase was inhibited by glucose 6-phosphate (Glc6P) with Ki values of 0.32 + 0.03 mm (C1I), 0.50 + 0.04 mm (MH 3924A) and -5 mM (brain). The inhibition could be abolished by 1 mM-AMP, indicating that AMP and Glc6P may partially compete for the same site on the protein. Liver phosphorylase a was not inhibited by up to 25 mM-Glc6P. In contrast with liver and brain isoenzymes, phosphorylase from the cell lines was not affected by NaF and Na2SO4. The data show that both the hepatocellular carcinoma and the non-malignant immortalized liver cells express a phosphorylase isoform different from the liver type. Furthermore, there is some evidence that the enzyme from MH 3924A and C1I cells is distinct from brain phosphorylase a, in spite of electrophoretic and immunological resemblance, and that this isoenzyme is subject to altered metabolic regulation.

Inhibition by 2‐Deoxyglucose and 1,5‐Gluconolactone of Glycogen Mobilization in Astroglia‐Rich Primary Cultures

“…The glycogen phosphorylase of this cell line is different from the liver enzyme and may well be identical with the fetal form, which very closely resembles the brain isoenzyme (for review, see Newgard et al, 1989). However, the Ki value of G6P for inhibition of phosphorylase a from rat brain was one order of magnitude higher than expected from this reasoning (Mayer et al, 1992). Thus, with the half-maximally inhibiting concentration of 0.8 mMfound in the present report, DG6P is a much more potent inhibitor of neural phosphorylase a than G6P.…”

Cytochemical and biochemical studies on adenylate cyclase activity in preneoplastic and neoplastic liver tissue and cultured liver cells

“…This may be the consequence of altered expression of glycogen phosphorylase isoenzymes in these cells. Whereas glycogenotic foci express liver-type glycogen phosphorylase, which, however, is present in the inactive form (Seelmann-Eggebert et al, 1987), C 1 I cells express a glycogen phosphorylase isoenzyme which is similar to the brain/tumor type and subject to different metabolic control (Mayer et al, 1992). The active form of this isoenzyme is inhibited by glucose 6-phosphate.…”

“…The loss in glycogen coincided with neoplastic transformation of the cells. The similarity in aberrations in glycogen metabolism between preneoplas-tic liver foci and the cell line renders the latter a suitable in vitro model for studying metabolic aberrations during neoplastic cell transformation (Mayer et al, 1992).…”

Glycogen Metabolic Genes Are Involved in Trehalose-6-Phosphate Synthase-Mediated Regulation of Pathogenicity by the Rice Blast Fungus Magnaporthe oryzae

“…Enzyme activities were determined spectrophotometrically in triplicate and are given as the concentration of product formed in one minute by total cell protein from 1 mg of mycelium. Amyloglucosidase activity [52] was determined by incubating 50 µl of each total cell protein solution with a 1% starch solution, excess purified hexokinase and excess purified glucose 6-phosphate dehydrogenase. Agl1 in the protein sample liberates glucose from starch, which is phosphorylated by hexokinase and the resulting glucose 6-phosphate used to generate NADPH from NADP using glucose 6-phosphate dehydrogenase.…”

“…The rate of increase in NADPH as the enzyme assay progressed, measured spectrophotometrically at A 340 and compared to the rate of NADPH production in assays containing no starch or boiled mycelial protein extract, was measured to calculate the activity of Agl1 in each sample. For glycogen phosphorylase activity [52], 50 µl of protein extract from each sample was added to 1 ml of enzyme assay, in triplicate. Each assay contained 4% glycogen, excess purified phosphoglucomutase and excess purified glucose 6-phosphate dehydrogenase.…”