PubMed · 179544
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R F Steiner, T Smith. 1976-04-19. Spin label studies on phosphorylase B.. https://doi.org/10.1016/0006-291x(76)90470-8
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The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.
The topological location of the nucleotide substrate binding environments on adenylate kinase has been explored with the fluorescent molecule [4-benzoyl]benzoyl-1-amidofluorescein (BzAF) and the nucleotide analog 3'-O-[4-benzoyl]benzoyl-ATP (BzATP), which are site-directed photoaffinity probes that bind covalently at the individual nucleotide sites. The MgBzATP substituted for MgATP as a substrate for this enzyme, whereas BzAF, which is neither a substrate nor a nucleotide, behaved as a competitive inhibitor for each nucleotide site independently. BzAF could be directed specifically to either of the nucleotide sites by near saturation of the second site with its natural nucleotide substrate. Using this second site blocking approach, each nucleotide site, in turn, could be protected competitively from BzAF-induced photoinhibition by the presence of its natural substrate. This strategy showed that under photolysis, the Kd(BzAF) of 0.1 mM (for the MgATP site) and 0.2 mM (for the AMP site) were nearly identical with the Km(app) values determined for MgATP and AMP, respectively. Pseudo first-order photolysis kinetics with [3H]BzAF revealed covalent binding stoichiometries for full inhibition of 1 mol of probe/mol of enzyme at either site. Thus, we prepared the binary complex ([3H]BzAF-enzyme) by photo-labeling the MgATP site-blocked enzyme with [3H]BzAf to 1:1 molar stoichiometry. Tryptic digestion followed by partial sequencing of the [3H]BzAF-labeled enzyme disclosed that BzAF was bound specifically within the peptide span Gly64-->Arg77. This amino acid domain therefore probably constitutes the neighborhood identifiable with AMP binding. Furthermore, we also achieved double photocovalent labeling of adenylate kinase with both photoprobes, by first cross-linking with approximately 1 mol of MgBzATP, followed by approximately 0.9 mol of [3H]BzAF, thus generating a ternary covalent complex. Comparison of the fluorescence of the binary species and the ternary (BzATP-enzyme-[3H]BzAF) complex revealed altered fluorescence emission profiles, which could indicate that a conformational change occurs during formation of the ternary (or transition state) complex.
Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required for DNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M(r) = 84,406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalian DNA ligase I and in Cdc17 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATP alpha S inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specificity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5' termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3' termini inhibited DNA ligation. The joining of DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.