Biomedical subjects

F Marcus

Publications and source records attributed to F Marcus.

At least 37 records · Page 2Linked to original sources

PubMed1988

Phosphorylation in situ of atrial natriuretic peptide prohormone at the cyclic AMP-dependent site.

Previously we have shown that atrial natriuretic peptides (ANP) are phosphorylated at Ser-104 by cyclic AMP-dependent protein kinase (Rittenhouse, J. Moberly, L., O'Donnell, M.E., Owen, N.E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610). In our present study, atrial natriuretic peptide prohormone (pro-ANP) purified from extracts of rat atria that had been incubated in situ with [32P]orthophosphate was found to be phosphorylated. The site of in situ phosphorylation was localized to the ANP region of the prohormone and was further delineated to a chymotryptic peptide having the same retention time by high performance liquid chromatography as the hexapeptide Arg101-Arg102-Ser103-Ser(P)104-Cys105-Phe106+ ++. This hexapeptide was also formed from in vitro phosphorylated pro-ANP and synthetic ANP. It is thus likely that Ser-104 is the site of phosphorylation of pro-ANP both in situ and in vitro. Incubation of atria with intracellular cyclic AMP-elevating agents consistently increased the specific radioactivity of the resulting purified pro-ANP by 5-10-fold. However, the overall extent of phosphorylation was low, suggesting that a subpopulation of pro-ANP molecules are phosphorylated at Ser-104 by a cyclic AMP-mediated pathway.

Amino Acid Sequence↗

PubMed1988

Comparative amino acid sequence of fructose-1,6-bisphosphatases: identification of a region unique to the light-regulated chloroplast enzyme.

Chloroplast fructose-1,6-bisphosphatase (Fru-P2-ase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from cytosolic gluconeogenic Fru-P2-ases. Light-dependent activation by way of a ferredoxin/thioredoxin system and insensitivity to AMP inhibition are distinctive characteristics of the chloroplast enzyme. However, the chloroplast enzyme shows a high degree of amino acid sequence similarity to gluconeogenic Fru-P2-ases. Sequence data reported for a total of 285 residues (approximately 75% of the structure) of the spinach chloroplast enzyme reveals a 46% amino acid sequence identity with pig kidney Fru-P2-ase. We now report the amino acid sequence of a region consisting of 46 additional residues. This region is located near the middle of the primary structure of the enzyme and it includes a 16-residue insert not present in other Fru-P2-ases. This sequence insert has two cysteines separated by only 4 amino acid residues (Cys-Val-Val-Asn-Val-Cys), a characteristic feature of at least three other enzymes containing redox-active cysteines. It appears likely that this region of chloroplast Fru-P2-ase is involved in light-dependent activation.

Amino Acid Sequence↗

PubMed1988

Amino-terminal sequence of spinach chloroplast fructose-1,6-bisphosphatase.

The sequence of the NH2-terminal 25-amino acid residues of purified spinach chloroplast fructose-1,6-bisphosphatase was determined by automated Edman degradation. The amino acid sequence is as follows: Ala-Ala-Val-Gly-Glu-Ala-Ala-Thr-Gln-Thr-Lys-Ala- Arg-Thr-Arg-Ser-Lys-Tyr-Glu-Ile-Glu-Thr-Leu-Thr-Gly. A comparison of this sequence with the corresponding region of pig kidney and yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatases shows that the sequence of residues 1-19 of the chloroplast enzyme has no homology with the other fructose-1,6-bisphosphatases, but homology is evident after residue 20. The dissimilar sequence contains a region (residues (8-17) rich in basic and hydroxylated amino acids, a structure which is typical of presequences of mitochondrial and chloroplast proteins. Since chloroplast fructose-1,6-bisphosphatase is nuclear in origin, these results suggest that the chloroplast targeting region may have been retained within the amino acid sequence of the mature protein.

Amino Acid Sequence↗

PubMed1987

Spinach chloroplast fructose-1,6-bisphosphatase: identification of the subtilisin-sensitive region and of conserved histidines.

Chloroplast fructose-1,6-bisphosphatase (FbPase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from those of cytosolic gluconeogenic FbPases. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. However, preliminary amino acid sequence data (78 residues) have demonstrated that a significant degree of amino acid sequence similarity exists between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase [Harrsch, P.B., Kim, Y., Fox, J.L., & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526]. In the present study, we have identified two structural features of spinach chloroplast FbPase that appear to be common to all FbPases. These include (a) the presence of a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FbPases and (b) the recognition of two conserved histidine residues, equivalent to histidines-253 and -311 of the mammalian enzymes. In addition, we have obtained sequence information accounting for more than three-fourths of the primary structure of spinach chloroplast FbPase. The high degree of homology observed between the chloroplast enzyme and gluconeogenic FbPases suggests a common evolutionary origin for all fructose-1,6-bisphosphatases in spite of their different functions and modes of regulation.

Chloroplasts↗

PubMed1987

Amino acid sequence similarity between malate dehydrogenases (NAD) and pea chloroplast malate dehydrogenase (NADP).

Purified pea chloroplast malate dehydrogenase (NADP) was reduced, S-pyridylethylated with 4-vinyl-pyridine and cleaved with trypsin. The resulting peptides were separated by reversed-phase high-performance liquid chromatography. Several of these peptides were subjected to automated Edman degradation. The sequences obtained were compared to the published primary structures of malate dehydrogenase from the thermophilic bacterium Thermus flavus and with the sequence of heart mitochondrial and cytoplasmic malate dehydrogenase (NAD). Most peptides from choroplast malate dehydrogenase (NADP) showed high homology with sequences of the other malate dehydrogenases, especially with those of the bacterial enzyme. One of the sequenced peptides contains the active-site histidine residue which is conserved in all malate dehydrogenases. Our results suggest a common evolutionary origin for all malate dehydrogenases despite their different coenzyme specificities and regulatory properties. The sequenced peptides which revealed no homology were either located at the amino-terminal or at the carboxy-terminal region of chloroplast malate dehydrogenase (NADP). These novel sequences are most likely plant-specific extensions of an ancestral malate dehydrogenase and may be responsible for the unique light-dependent activation of the chloroplast enzyme.

Amino Acid Sequence↗

PubMed1987

Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site.

In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of [32P] orthophosphate. Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography. Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939-3943). The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11. We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests. Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit. However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures. An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures. The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.

Amino Acid Sequence↗

PubMed1987

A quantitative bioassay to measure the rate of respiration of isolated rat tooth pulp tissue.

The formation of carbon dioxide, expressed as milligrammes of carbon dioxide per gramme wet weight, was used to determine the rate of succinate-induced respiration in rat incisal dental pulp, after incubation in a phosphate buffer. Seven experimental parameters: (a) physical nature of the tissue; (b) mass of the rat; (c) pre-incubation time; (d) incubation time; (e) substrate concentration; (f) effect of pH; and (g) effects of technique on pH, were analysed; pH was a critical determinant for success with this assay.

Animals↗

PubMed1987

Function, structure and evolution of fructose-1,6-bisphosphatase.

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 this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Amino Acid Sequence↗

PubMed1986

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.

Amino Acid Sequence↗

PubMed1986

Phosphorylation of atrial natriuretic peptides by cyclic AMP-dependent protein kinase.

Atrial natriuretic peptides refer to a family of related peptides secreted by atria that appear to have an important role in the control of blood pressure. The structure of these peptides shows the amino acid sequence Arg101-Arg102-Ser103-Ser104, which is a typical recognition sequence (Arg-Arg-X-Ser) for phosphorylation by cyclic AMP-dependent protein kinase. With this background, we tested two synthetic atrial natriuretic peptides (Arg101-Tyr126 and Gly96-Tyr126) as substrates for in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. The tested atrial natriuretic peptides were found to be substrates for the reaction. Sequence studies demonstrated that the site of phosphorylation was located, as expected, at Ser104. Kinetic studies demonstrate that both atrial natriuretic peptides are excellent substrates for cyclic AMP-dependent protein kinase. In particular, the longer peptide Gly96-Tyr126 exhibited an apparent Km value of about 0.5 microM, to our knowledge the lowest reported Km for a cyclic AMP-dependent protein kinase substrate. Preliminary studies to measure the biological activity of the in vitro phosphorylated atrial peptides indicate that these compounds are more effective than the corresponding dephospho forms in stimulating Na/K/Cl cotransport in cultured vascular smooth muscle cells.

Amino Acid Sequence↗

PubMed1986

Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.

Amino Acid Sequence↗

PubMed1986

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.

Amino Acid Sequence↗

PubMed1985

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.

Amino Acid Sequence↗

PubMed1985

Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein.

Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.

Adenosine Monophosphate↗

PubMed1985

Distribution of a COOH-terminal amino acid extension of liver fructose-1,6-bisphosphatase among rodent species.

We have recently established from sequence analysis that rat liver fructose-1,6-bisphosphatase contains a 24-26 residue extension beyond the COOH-terminal amino acid of other mammalian fructose-1,6-bisphosphatases that results in an increased subunit molecular weight (Rittenhouse et al. (1983) J. Biol. Chem. 258, 7648-7652). In the present work the distribution of the COOH-terminal extension of fructose-1,6-bisphosphatases was tested by subunit molecular weight analysis of the enzyme immunoprecipitated from liver extracts. Of all rodent species tested, including several Muridae other than Rattus; only the enzyme from animals of the genus Rattus was found to have the extension. Further studies on the distribution of the enzyme extension could provide a simple tool to study the phylogeny of the genus Rattus.

Amino Acid Sequence↗

PubMed1985

Preferential cleavage at aspartyl-prolyl peptide bonds in dilute acid.

A simple, rapid technique is presented for preferential cleavage at aspartylprolyl peptide bonds. The method is based upon the fact that these peptide bonds are 8-20-fold more labile in 0.015 N HCl at 100-110 degrees than other aspartyl-X or X-aspartyl peptide bonds. The method has proven effective in the cleavage of several peptides from pig kidney fructose-1,6-bisphosphatase and should facilitate sequence analysis of proteins that contain aspartyl-prolyl linkages.

Amino Acid Sequence↗

PubMed1984

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase.

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.

Animals↗

PubMed1984

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.

Animals↗