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F Marcus

Publications and source records attributed to F Marcus.

At least 55 records · Page 3Linked to original sources
PubMed1984

A re-evaluation of the molecular weight of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase.

In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S. cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide. Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S. cerevisiae. The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.

Fructose-Bisphosphatase↗
PubMed1984

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 microM, 50% inhibition was observed at 4.8 microM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (Ki = 16 microM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate - fructose-1,6-bisphosphatase active site interaction.

Adenosine Monophosphate↗
PubMed1984

Identification of the in vivo and in vitro phosphorylation sites of rat liver fructose 1,6-bisphosphatase.

Rat liver fructose 1,6-bisphosphatase appears to be unique in that it extends 24-26 residues beyond the COOH-terminal amino acid of other mammalian fructose 1,6-bisphosphatases and this extension contains phosphorylation sites. Using as a frame of reference the 335-residue sequence of pig kidney fructose 1,6-bisphosphatase (Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165), the rat liver enzyme would extend to residue 361. Limited proteolysis in the COOH-terminal region of the molecule with chymotrypsin, trypsin, or both sequentially, led us to establish that the phosphorylation sites are located at Ser residues 341 and 356. The in vitro phosphorylation of purified rat liver fructose 1,6-bisphosphatase by the catalytic subunit of cyclic AMP-dependent protein kinase results in modification at both residues, although the major site of phosphorylation (61%) is at Ser-341. In contrast, rat liver fructose 1,6-bisphosphatase purified from animals that had been injected with [32P] phosphate contains most of the label (81%) at Ser-356.

Amino Acid Sequence↗
PubMed1984

Identification of the highly reactive sulfhydryl group of pig kidney fructose 1,6-bisphosphatase at cysteine 128.

Fructose 1,6-bisphosphatases contain a highly reactive cysteine residue, the reactivity of which is influenced by ligands that bind at the catalytic and at the allosteric AMP sites of the enzyme. Nevertheless, the sulfhydryl group appears to be proximal to these sites and not a functional component of either. Modification of pig kidney fructose 1,6-bisphosphatase with three reagents, 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide, and phenacyl bromide, yields derivatives with similar properties, thus suggesting that the same residue was modified in each case. The modified enzymes exhibited: (a) higher Vmax when Mn2+ was used as the activating cation; (b) decreased activity in the presence of nonsaturating Mg2+ concentrations; (c) no change in sensitivity toward AMP inhibition. Automated Edman degradation of a tryptic peptide containing radioactive carboxamidomethylcysteine showed the sequence of residues Gly-111-Arg-140 of pig kidney fructose 1,6-bisphosphatase. The modified residue was shown to be cysteine-128, and the same cysteine residue was alkylated when the enzyme was reacted with phenacyl bromide. Cysteine-128 is also present in rat and sheep liver fructose 1,6-bisphosphatase and a long stretch of the sequence around this reactive cysteine residue is highly conserved.

Adenosine Monophosphate↗
PubMed1984

Comparison of a QRS scoring system for estimating acute infarct size with radionuclide left ventriculography.

A QRS scoring system was compared with left ventricular ejection fraction (LVEF) in 40 patients enrolled in the Multicenter Post Infarction Program. A poor correlation was found between these two parameters. Possible reasons for these findings include the fact that the radionuclide studies were performed at several institutions or that there was a mean interval of 6 days between the time of the ECG and the radionuclide studies. It was determined that the ECGs could be scored by inexperienced scorers. The utility and limitation of the QRS scoring system for prediction of LVEF need further evaluation, particularly if it is to be applied to a multicenter study.

Cardiac Output↗
PubMed1983

Amino acid sequence of the COOH-terminal region of fructose-1,6-bisphosphatases in relation to cyclic AMP-dependent phosphorylation.

Studies of in vitro phosphorylation of four different gluconeogenic fructose-1,6-bisphosphatases by the catalytic subunit of cyclic AMP-dependent protein kinase have shown that only rat liver fructose-1,6-bisphosphatase is a substrate of the protein kinase. A comparison of the molecular weights of fructose-1,6-bisphosphatases revealed that the nonphosphorylatable mouse liver, rabbit liver, and pig kidney enzymes have a subunit Mr approximately 37,000 while the subunit molecular weight of purified rat liver fructose-1,6-bisphosphatase is about 41,000 (Hosey, M. M., and Marcus, F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 91-94). To probe the structural basis for the higher molecular weight and unique phosphorylation of rat liver fructose-1,6-bisphosphatase, the CNBr fragment containing the phosphorylation site was purified and the amino acid sequence of this 43-residue peptide was determined. The sequence data revealed that the rat liver enzyme extends 24-26 residues beyond the COOH-terminal amino acid of pig kidney and rabbit liver fructose-1,6-bisphosphatase and that cyclic AMP-dependent phosphorylation sites are located in this proline-rich extension. The kinetic properties of rat liver fructose-1,6-bisphosphatase do not appear to be influenced in any way, either by the COOH-terminal extension itself or by the state of phosphorylation. Polyacrylamide gel electrophoresis of immunoprecipitates from crude extract supernatants demonstrated that the rat liver enzyme is larger than other fructose-1,6-bisphosphatases studied to date, and that the differences in molecular weight are not due to proteolytic modification of other fructose-1,6-bisphosphatases during isolation procedures.

Amino Acid Sequence↗
PubMed1983

Failure to secrete immunoreactive insulin by rats fed a low protein diet.

Rats fed a 6% protein diet for 14 weeks showed a normal glucose tolerance but failed to release immunoreactive insulin (IRI), following iv glucose. Furthermore the isolated perfused pancreas of the rats fed the 6% protein diet secreted eleven times less IRI than the isolated pancreas prepared from the rats reared on an 18% protein diet when both were subjected to the same glucose challenge. This difference occurred despite the fact that the total IRI content of the pancreas' was not significantly different between the two groups. In the protein-malnourished rats it is suggested that the normal glucose tolerance response, despite the failure to release a concommitant amount of IRI, may indicate increased sensitivity to IRI or may be due to the secretion in these animals of a biologically active insulin, which shows no immunological cross-reactivity with pancreatic insulin. Alternately there may be increased peripheral glucose utilisation in the low protein rats.

Animals↗
PubMed1982

A method for determining kinetic parameters at high enzyme concentrations.

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

Adenosine Monophosphate↗
PubMed1982

Complete amino acid sequence of pig kidney fructose-1,6-bisphosphatase.

The covalent structure of the pig kidney fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) subunit has been determined. Placement of the 335 amino acid residues in the polypeptide chain was based largely on automated Edman degradation of eight purified cyanogen bromide fragments generated from the S-carboxymethylated protein. The determination of the amino acid sequence of the largest cyanogen bromide fragment (154 residues) required additional analysis of subfragments obtained by tryptic cleavage at arginyl residues and by mild acid cleavage of an Asp-Pro peptide bond. Alignment of the cyanogen bromide fragments was accomplished by analysis of a product of limited proteolysis by an endogenous protease and by characterization of the tryptic peptides isolated from S-[14C]carboxymethylated fructose-1,6-bisphosphatase. This sequence information has permitted the identification of several reactive sites of functional and structural significance in pig kidney fructose-1,6-bisphosphatase.

Amino Acid Sequence↗
PubMed1981

The covalent structure of pig kidney fructose-1,6-bisphosphatase. Sequence of a 63-residue cyanogen bromide peptide containing a phosphorylatable serine.

Native pig kidney fructose-1,6-bisphosphatase, in contrast to the rat liver enzyme, is not a substrate of cyclic AMP-dependent protein kinase. However, the pig kidney enzyme becomes a substrate when phosphorylation is performed in 1.6 M urea, after prior unfolding in 8 M urea. A cyanogen bromide fragment containing the phosphorylation site has been isolated and the amino acid sequence of this 63-residue peptide has been determined. This peptide has the following sequence: Leu-Asp-Pro-Ala-Ile-Gly-Glu-Phe-Ile-Leu-Val-Asp-Arg-Asn-Val-Lys-le-Lys-Lys-Lys- Gly-Ser(P)-Ile-Tyr-Ser-Ile-Asn-Glu-Gly-Tyr-Ala-Lys-Glu-Phe-Asp-Pro-Ala-Ile-Thr- Glu-Tyr-Ile-Glu-Arg-Lys-Lys-Phe-Pro-Pro-Asp-Asn-Ser-Ala-Pro-Tyr-Gly-Ala-Arg-Tyr -Val-Gly-Ser-Met. The amino acid sequence around the phosphorylated serine residue resembles those of other protein substrates of cyclic AMP-dependent protein kinase, but it is completely different from the phosphorylation site found in native rat liver fructose-1,6-bisphosphatase.

Amino Acid Sequence↗
PubMed1981

Mouse (C57BL/KsJ) liver phosphofructokinase. Allosteric kinetics and age-related changes in the genetically diabetic state.

The regulatory kinetic properties of phosphofructokinase partially purified from the livers of C57BL/KsJ mice were studied. The fructose 6-phosphate saturation curves were highly pH dependent. At a fixed MgATP concentration (1 mM), allosteric kinetics was observed in the range of pH studied (7.3 to 8.3) and the S0.5 values for fructose 6-phosphate decreased by about 0.2 to 0.3 mM for each 0.1-unit increment in pH. Allosteric effects on the sigmoidal response to fructose 6-phosphate: activation by AMP, NH4+, and glucose 1,6-bisphosphate, inhibition by MgATP2-, and synergistic inhibition between ATP and citrate, were all present at pH 8.0 to 8.2. Comparative kinetic studies with liver phosphofructokinase isolated from both the normal (C57BL/KsJ) and the genetically diabetic (C57BL/KsJ-db) mice of 9 to 10 and 15 to 16 weeks of age showed that the enzyme from the livers of diabetic mice exhibited decreased activity at subsaturating concentrations of fructose 6-phosphate. However, phosphofructokinase isolated from the livers of normal and genetically diabetic mice of 4 to 5 weeks of age showed no difference in kinetic properties. Thus, there appears to be a correlation between the change in properties of liver phosphofructokinase and the expression of hyperglycemia and obesity in the genetically diabetic mice. The decreased activity of liver phosphofructokinase in the older diabetic animals may well be one of the causes of the increased blood glucose levels. The results are also discussed in a general context with regard to the possible role of phosphofructokinase in the regulation of hepatic gluconeogenesis.

Aging↗
PubMed1981

Fructose-bisphosphatase as a substrate of cyclic AMP-dependent protein kinase.

We have tested rat liver fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) and three other gluconeogenic fructose-bisphosphatases as substrates for the catalytic subunit of cyclic AMP-dependent protein kinase. In contrast to the rat liver enzyme, homogeneous preparations of mouse liver, rabbit liver, and pig kidney fructose-bisphosphatase could not be phosphorylated by the kinase. Comparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the four above fructose-bisphosphatases revealed that the subunit molecular weight of the isolated rat liver enzyme (ca. 40,000-42,000) was greater than that of mouse liver, rabbit liver, and pig kidney fructose-bisphosphatases (ca. 36,000-37,000). Treatment of 32P-labeled rat liver fructose-bisphosphatase with trypsin resulted in the conversion of the rat liver enzyme to an active species with a subunit molecular weight identical to that of the three other enzymes, with complete loss of the 32P-labeled site. Identical trypsin treatment of pig kidney fructose-bisphosphatase caused no change in the molecular weight of the enzyme. The results suggest that the purified mouse liver, rabbit liver, and pig kidney fructose-bisphosphatases are not substrates for the cyclic AMP-dependent protein kinase in vitro because they lack the phosphorylation-site peptide.

Animals↗