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M Stefani

Publications and source records attributed to M Stefani.

At least 73 records · Page 4Linked to original sources
PubMed1989

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.

Acid Anhydride Hydrolases↗
PubMed1989

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase.

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.

Acid Phosphatase↗
PubMed1988

CNS metastasis in ovarian cancer with microangiopathic hemolytic anemia associated with diffuse intravascular coagulation.

We present the case of a woman affected by ovarian cancer metastatic to multiple lymph node and the CNS. She was affected by hemorrhagic diathesis with microangiopathic alterations, whereas coagulopathy developed only after some days in coincidence with disease worsening. Our patient is probably one of those in which cancer leads to microangiopathy and coagulopathy by means of a tissue factor-like activity, a common event in mucin secretory tumors. Fibrinolytic activity was also increased in our patient as in others of the same type. The main aspect of this case report is metastasis to the CNS and to other multiple sites, which is quite uncommon in such cancers. We retain that tumor procoagulant activity could have played a role in this phenomenon.

Adenocarcinoma, Papillary↗
PubMed1988

Horse brain acylphosphatase: purification and characterization.

Two structurally different acylphosphatases found in horse brain were purified; they were not immunologically related. The molecular masses were almost identical and the kinetic parameters were rather similar. The data reported indicate that one of the purified brain acylphosphatases and an enzyme, previously isolated from horse muscle, are the same protein. The presence of this acylphosphatase form in the brain has not been reported before. The other acylphosphatase seemed to be the same as the enzyme which had been purified from calf brain and partially characterized by Diederich and Grisolia [(1969) J. Biol. Chem. 244, 2412-2417]. Furthermore, this enzyme seems to be identical to the acylphosphatase recently purified in our laboratory from human erythrocytes.

Acid Anhydride Hydrolases↗
PubMed1988

Effect of exogenously added acylphosphatases on inositol lipid metabolism in human platelets.

In this paper we demonstrate that human platelets contain an acylphosphatase isoenzyme. We then investigated the effect of exogenously added human muscle and erythrocyte acylphosphatases on inositol lipid content in human platelets permeabilized with saponin. Alterations in the level of the polyphosphoinositides were observed: in particular, the levels of phosphatidylinositol 4,5-bisphosphate, and of phosphatidylinositol 4-monophosphate were decreased, whereas the level of phosphatidylinositol was increased. These results suggest that acylphosphatases promote polyphosphoinositide dephosphorylation, possibly through intracellular Ca2+ mobilization.

Acid Anhydride Hydrolases↗
PubMed1988

Guinea pig acylphosphatase: the amino acid sequence.

We determined the primary structure of guinea pig skeletal muscle acylphosphatase, using the high degree of homology with several vertebrate acylphosphatases to obtain correct alignment of the complete series of tryptic peptides. Their sequences were obtained mainly by Edman degradation; FAB mass spectrometry was used to identify the acyl group blocking the NH2-terminal residue and to elucidate the structure of the NH2-terminal tryptic peptide. The comparison among acylphosphatase sequences from skeletal muscle of several vertebrate species is presented and discussed.

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↗
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↗