Frank Marcus

Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers☆

Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.

Phosphorylation of muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase

Abstract The catalytic subunit of cyclic AMP-dependent protein kinase catalyzes the phosphorylation of rabbit skeletal muscle phosphofructokinase. The reaction is inhibited by the specific inhibitor of protein kinase and proceeds at about 2% the rate observed with phosphorylase kinase but more rapidly than with rat liver fructose bisphosphatase as substrate. Maximum extent of incorporation (0.43 to 0.85 moles per mole of protomer) plus the covalently-bound phosphate present in the isolated enzyme (0.20 to 0.34 moles per mole) approaches one mole per mole.

[59] Fructose-1,6-bisphosphatase from rat liver

Publisher Summary This chapter describes the purification and properties of the enzyme fructose-l,6-bisphosphatase obtained from rat liver. Fructose-l,6-bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and P i . The enzyme activity can be determined spectrophotometrically by following the rate of formation of NADPH at 340 nm in the presence of excess glucosephosphate isomerase and glucose-6-phosphate dehydrogenase. The purification of the enzyme involves extraction, heat treatment, phosphocellulose AffiGel blue chromatography, ammonium sulfate fractionation, and AffiGel blue chromatography. Rat liver fructose-1,6-bisphosphatase exhibits a single protein band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis with a subunit molecular weight of 42,000. Purified rat liver fructose-1,6-bisphosphatase and the enzyme present in crude extracts shows maximum activity at neutral pH. Fructose-1,6-bisphosphatase requires a divalent cation for activity and this requirement can be fulfilled either by Mg 2+ or Mn 2+ . Zn 2+ , Fe 2+ , and Fe 3+ are strongly inhibitory to the enzyme.

Amino acid sequence homology among fructose-1,6-bisphosphatases

The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

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.

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Abstract Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (Mr ca. 29,000), and the S-peptide (Mr 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH2-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH2-terminal 60-amino acid peptide.

Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.

Reactivity of the thiol groups of Escherichia coli phosphofructo-1-kinase

Modification of Escherichia coli phosphofructo-1-kinase (6-phosphofructokinase; EC 2.7.1.11) with several thiol modifying reagents led to a pseudo-first-order loss of activity that was associated with the modification of a single cysteine residue, identified as the cysteine at position 119 in the protein sequence. This cysteine was protected from reaction with vinyl pyridine, bromopyruvate, and dithionitrobenzoic acid by the substrate, fructose-6-P. In the crystal structure of the highly homologous phosphofructokinase from Bacillus stearothermophilus, cysteine 119 is sufficiently distant from the catalytic site to exclude a direct steric inhibition of the binding of substrate as a mechanism of inactivation for the modification. Thus, the inhibition is unlikely to be a direct one but to be the result of interference with the conformational change that is associated with fructose-6-P binding. A second thiol, position 283, was shown to be protected from reaction when the enzyme was in its native conformation. In contrast to the previously published sequence for the E. coli enzyme six cysteines as opposed to seven have been found both in enzyme from strain LE392 and in enzyme produced by a plasmid that was derived from pLC 16-4. The position in question, 75, was identified as phenylalanine.

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 bakers yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of bakers 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.

Amino acid sequence homology between yeast hexokinases and rat hexokinase C.

Automated Edman degradation of seven purified tryptic peptides from Novikoff hepatoma hexokinase C revealed amino acid sequences that could be easily aligned within the primary structure of yeast hexokinases. This high degree of structural homology suggests a common evolutionary origin for mammalian and yeast hexokinases. Some of the sequenced peptides overlapped with each other, as well as with regions of the sequence of yeast hexokinases, suggesting that during evolution the 100,000 molecular weight subunit mammalian hexokinases may have resulted from gene duplication followed by gene fusion from a pre-vertebrate 50,000 molecular weight hexokinase ancestor.