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G Ramponi

Publications and source records attributed to G Ramponi.

At least 163 records · Page 9Linked to original sources
PubMed1988

Secondary structure of acylphosphatase from rabbit skeletal muscle. A nuclear magnetic resonance study.

Sequence-specific assignment of 1H nuclear magnetic resonance spectra of acylphosphatase (EC 3.6.1.7) isolated from rabbit skeletal muscle have made it possible to identify short distance constraints from nuclear Overhauser enhancement spectra, to evaluate spin-spin coupling constants of many backbone amide hydrogens and to assess their slow exchange with deuterons in 2H2O solution. Analysis of these data show that the major regular secondary structure of the enzyme consists of five extended beta-strands, four of which are arranged in an antiparallel beta-sheet, while the fifth is attached parallel. A helix consisting of 11 residues has also been identified. Consideration of additional distance constraints between sequentially remote residues has allowed us to give an outline of the overall fold of the protein.

Acid Anhydride Hydrolases↗
PubMed1988

Acylphosphatase increases the rate of ethanol production from glucose in cell-free extracts of Saccharomyces cerevisiae.

Addition of acylphosphatase exerted a stimulating effect on the alcoholic fermentation of glucose by Saccharomyces cerevisiae. The rates of glucose degradation and ethanol production by cell-free extracts of the S-288C strain were measured in the absence and in the presence of various levels of this enzyme. Two acylphosphatase isoenzymes were used; one was purified from horse skeletal muscle and the other from human erythrocytes. Both increased the rate of alcoholic fermentation, but that from erythrocytes proved to be the more efficient. This stimulating action is probably due to an "uncoupling effect" of acylphosphatase on the fermentative process, through hydrolysis of 3-phosphoglyceroyl phosphate. This was demonstrated by the fact that alcoholic fermentation was stimulated considerably by a mixture of ADP and inorganic phosphate and by arsenate as well. The possibility of improving the fermentative capacity of microorganisms may have important biotechnological applications.

Acid Anhydride Hydrolases↗
PubMed1988

Partial purification and biochemical characterization of human plasma thrombopoietin.

A factor that stimulates the incorporation of 75Selenomethionine into the newly formed platelets of recipient mice (thrombopoietin, TPO) has been partially purified from the plasma of thrombocytopenic patients. The activity was precipitated at 60-80% ammonium sulfate saturation and further purified with hydrophobic interaction chromatography. Thrombopoietin was retained by concanavalin-A-Sepharose. Using HPLC size-exclusion chromatography, an approximate molecular weight of 40,000 dalton was calculated. The overall purification factor was about 2,100-fold. TPO was stable in a pH range from 5 to 9 and was heat-sensitive, and the biological activity was destroyed by trypsin treatment and by dithiothreitol. The partially purified molecule did not stimulate the proliferation of megakaryocyte progenitors in vitro and had no effect on the growth of erythroid or granulocyte-macrophage colonies; when administered in-vivo, TPO significantly affected the mean platelet volume and increased the number of small acetylcholinesterase cells in the bone marrow. TPO appears to be specific for the megakaryocytic lineage and active on the postmitotic compartment of megakaryocytes.

Glycoproteins↗
PubMed1987

Acylphosphatase levels of human erythrocytes during cell ageing.

Human erythrocytes contain an acylphosphatase isoenzyme, whose concentration during cell ageing was determined by an enzyme immunoassay. Erythrocytes were age-fractionated by isopycnic centrifugation in "Percoll" density gradients. Acylphosphatase concentration was found to rise with the increase of cell density. Maximum values were attained in the mature erythrocytes and there was only a slight decrease in the older cells. Acylphosphatase activity in human erythrocytes of different ages followed a similar pattern, a finding which was confirmed for rabbit red cells with different levels of reticulocytes. The most probable mechanism for the increase of acylphosphatase content and activity during red cell maturation appears to be a de novo synthesis of this enzyme during the reticulocyte stage and its storage, virtually unaffected, in the mature erythrocytes. The possible consequences of increased acylphosphatase levels on the metabolic modifications associated with erythrocyte ageing are discussed.

Acid Anhydride Hydrolases↗
PubMed1986

A new acylphosphatase isoenzyme from human erythrocytes: purification, characterization, and primary structure.

A new acylphosphatase from human erythrocytes was isolated by an original purification procedure. It is an isoenzyme of the well-characterized human skeletal muscle acylphosphatase. The erythrocyte enzyme shows hydrolytic activity on acyl phosphates with higher affinity than the muscle enzyme for some substrates and phosphorylated inhibitors. The sequence was determined by characterizing the peptides purified from tryptic, peptic, and Staphylococcus aureus V8 protease digests of the protein, and it was found to differ in 44% of the total positions as compared to the human muscle enzyme. About one-third of these differences are in the form of strictly conservative replacements. The protein consists of 98 amino acid residues; it has an acetylated NH2-terminus and does not contain cysteine: (sequence in text).

Acid Anhydride Hydrolases↗
PubMed1986

Purification and characterization of rabbit muscle acylphosphatase in the thiol (-SH) form.

Modifications in the muscle acylphosphatase purification procedure enabled us to isolate the enzyme with its sole cysteine in the -SH form; this enzyme form is the most abundant in vivo. Our data demonstrates that the enzyme forms purified by previously reported procedures can be easily derived from a reaction of the SH-enzyme with oxidized glutathione. Probably most, or even all, of these enzyme forms are artifacts due to the purification. The SH-acylphosphatase shows kinetic parameters similar to those reported for the mixed disulfide with glutathione and S-S dimer, except for the specific activity value, which is about twice as much, and the Km, which is reduced.

Acid Anhydride Hydrolases↗
PubMed1985

Rabbit skeletal muscle acylphosphatase: the amino acid sequence of form Ra1.

Acylphosphatase was purified from rabbit skeletal muscle by a procedure involving an affinity chromatography step on immunoadsorbent and subsequent ion-exchange chromatography. Three molecular forms with acylphosphatase activity, named Ra1, Ra2, and Ra3, were purified and characterized with respect to molecular weight, amino acid composition, and main kinetic parameters. The amino acid sequence of Ra1 is given in the present paper. The Ra1 form consists of a single polypeptide chain of 98 amino acid residues and contains only one cysteine residue at position 21 that is S-S bound to glutathione. The polypeptide chain has an acetyl group blocking the NH2 terminus. Ra1, Ra2, and Ra3 are compared with the corresponding molecular forms isolated from skeletal muscle of horse and turkey.

Acid Anhydride Hydrolases↗
PubMed1985

Acylphosphatase from human skeletal muscle: purification, some properties and levels in normal and myopathic muscles.

Human skeletal muscle acylphosphatase was purified by immunoaffinity chromatography using anti-horse muscle acylphosphatase antibodies. The three forms of the enzyme present in human muscle are very similar to those found in muscles of other animal species. The two main forms, Hu 1 and Hu 3, were also characterized with respect to molecular weight and some kinetic properties. Levels of acylphosphatase activity were measured in specimens of muscle from normals and from patients with various forms of muscular dystrophies and other myopathies. Acylphosphatase activity appears to be lower in all myopathic forms considered than in controls, and seems to be correlated with percentage of Ca2+ activation of (Ca2+ + Mg2+)-ATPase.

Acid Anhydride Hydrolases↗
PubMed1984

Studies on synthesis and degradation rates and some molecular properties of guinea-pig muscle acylphosphatase.

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) was purified from guinea-pig muscle by a procedure involving immuno-affinity chromatography and a subsequent ion-exchange chromatography. This purification technique gave an overall yield of about 60% and permitted the isolation of three molecular forms with acylphosphatase activity, with a distribution greatly resembling those found in horse and turkey muscle. The main form appears to be very similar to the corresponding form in horse and turkey muscle, as indicated by amino acid composition, end-group analysis, the presence of glutathione as a mixed disulphide in almost the same stoichiometric ratio and kinetic analysis. From turnover data, the main form of acylphosphatase in guinea-pig muscle exhibits a degradation constant of 0.10 day-1, corresponding to a half-life of 6.8 days. These values are very close to those found for muscle total soluble proteins.

Acid Anhydride Hydrolases↗
PubMed1984

Human skeletal muscle acylphosphatase: the primary structure.

Human skeletal muscle acylphosphatase, purified by a technique based on affinity chromatography on immunoadsorbent, has been sequenced completely using tryptic and peptic peptide series, prepared by reverse-phase high-pressure liquid chromatography. The sequence analysis was carried out on all the isolated tryptic peptides using a manual Edman degradation technique and time-course analysis of the released amino acids by carboxypeptidase A. The enzyme is NH2-blocked and the blocking group has been identified by fast atom bombardment mass spectrometry.

Acid Anhydride Hydrolases↗
PubMed1983

The primary structure of turkey muscle acylphosphatase.

The complete primary structure of turkey muscle acylphosphatase has been determined. The sequence was derived from peptides obtained by digestion of the carboxymethylated protein with pepsin and thermolysin and by subdigestion of some of the cyanogen bromide fragments with trypsin and Staphylococcus aureus protease. Peptides were purified by preparative finger prints and/or preparative high-performance liquid chromatography. Sequencing of the various peptides was achieved by manual Edman degradation and by time-course analysis of amino acids released by carboxypeptidases. The amino-terminal blocking group (acetyl) was determined by fast atom bombardment mass spectrometry. This sequence was compared with that of horse muscle enzyme determined previously.

Acid Anhydride Hydrolases↗
PubMed1983

Affinity chromatographic purification of horse muscle acylphosphatase: evidence of the existence of multiple molecular forms.

Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enzyme is a mixed disulfide between a main chain of 98 amino acid residues and glutathione. Ho2 differs from Ho1 only in the chemical nature of the molecule(s) S-S bound to the sole cysteine present at position 21 of the main chain. Ho3 is an S-S dimer of the main polypeptide chain. Ho1, Ho2, and Ho3 elicit very similar kinetic parameters in the presence of benzoylphosphate as a substrate.

Acid Anhydride Hydrolases↗
PubMed1982

Purification of horse muscle acylphosphatase antibodies by affinity chromatography.

Horse muscle acylphosphatase antibodies were obtained by immunizing rabbits with the highly purified antigen cross-linked with glutaraldehyde. Specific antibodies were purified from the immunoglobulin fraction by affinity chromatography using a matrix coupled with the pure antigen as immunoadsorbent. The purified antibodies were partially characterized by immunodiffusion and immunoprecipitin techniques. These antibodies could be used to study aspects of the muscle acylphosphatase structure, localization and other biological properties.

Acid Anhydride Hydrolases↗
PubMed1981

Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological cross-reactivity with the rat liver enzyme.

The purification of mitochondrial carbamoyl phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) from small samples of human liver is described. The enzyme is composed of a single polypeptide of Mr 160 000 +/- 500 as shown by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents. The synthetase migrates in polyacrylamide gradient gels in the absence of detergents at a rate corresponding to a Mr of 165 000. Estimates of the molecular weight of the native enzyme by gel filtration and density gradient centrifugation yield a value of 178 000. The results indicate that the enzyme exists predominantly as monomeres. Amino acids composition, isoelectric point, stability, Km values and the ability to catalyze partial reactions have been measured and compared with known properties of carbamoyl phosphate synthetases from other sources. From the available data a high degree of evolutionary conservation of the ammonia-dependent synthetase is suggested. This is also supported by the demonstration of extensive immunological cross-reactivity between the human and rat enzymes.

Amino Acids↗