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P Fasella

Publications and source records attributed to P Fasella.

At least 37 records · Page 2Linked to original sources
PubMed1979

Catalytic activity of aspartate aminotransferase in the crystal. Equilibrium and kinetic analysis.

Natural substrates and analogs rapidly diffuse through crystals of pig heart mitochondrial aspartate aminotransferase and react at the active sites causing spectral changes that can be measured by single-crystal microspectrophotometry. Dissociation constants for natural substrates and rate constants of transamination for slowly reacting substrates have been determined. A comparison between the data obtained in the crystal and in solution shows that the crystalline enzyme is catalytically competent and that events occurring in the crystal essentially parallel those occurring in solution, even though minor differences have been detected.

Animals↗
PubMed1979

A collagen film for microdetermination of collagenase activity.

A simple, rapid, sensitive, and specific film assay for collagenase activity employing a glass-supported, reconstituted collagen gel is described. Digestion of the collagen film results in sharply defined zones of lysis detectable by staining with Coomassie blue. The assay is semiquantitative and suitable for micro enzyme determination in biological fluids.

Microbial Collagenase↗
PubMed1978

Physico-chemical evidence for the interaction between aldolase and glyceraldehyde-3-phosphate dehydrogenase.

Polarization of fluorescence measurements of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocyanate have been used to detect the possible formation of a soluble complex between the proteins. The results suggest an interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase with an apparent dissociation constant 3 X 10(-7) M and an apparent stoichiometry of two aldolase tetramers bound per tetramer of D-glyceraldehyde-3-phosphate dehydrogenase.

Fluoresceins↗
PubMed1978

Infrared stretching frequencies of CO in carbomonoxyhemoglobin from trout.

The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.

Animals↗
PubMed1976

An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart.

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.

Amino Acid Sequence↗
PubMed1976

Kinetics of the interaction between aspartic aminotransferase and anions.

The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature-jump technique. The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions. A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 mus with phosphate, 20-100 mus with cacodylate and 1-2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a chi2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.

Animals↗
PubMed1976

Serine transhydroxymethylase from rabbit liver. Sequence of anonapeptide at the pyridoxal-5'-phosphate-binding site.

The amino acid sequence of the coenzyme-binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBH4 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated. A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme-binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val-Val-Thr-Thr-His(Pxy)-Thr-Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins.

Amino Acid Sequence↗
PubMed1976

The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered.

Animals↗
PubMed1975

The primary structure of aspartate aminotransferase from pig heart muscle. Digestion with a proteinase having specificity for lysine residues.

Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.

Amino Acid Sequence↗
PubMed1973

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase.

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

Amino Acid Sequence↗