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Biomedical subjects

P M Horowitz

Publications and source records attributed to P M Horowitz.

At least 145 records · Page 8Linked to original sources
PubMed1984

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking.

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.

Electrophoresis, Agar Gel↗
PubMed1983

Binding of colchicine to renal tubulin at 5 degrees C.

Previous reports in the literature state that the binding of colchicine to soluble tubulin is negligible at 0 - 4 degrees C as measured by non-equilibrium binding methods. In contrast, we have detected significant binding of colchicine to tubulin at 5 degrees C. Furthermore, for the first time equilibrium dialysis has been used to measure colchicine binding. The value of the dissociation constant was 1.8 microM at 5 degrees C, and the stoichiometry of colchicine binding at 5 degrees C equaled that at 37 degrees C. Dissociation at 5 degrees C of bound colchicine was negligible over a period of 23 h, and the estimated minimal half-time of dissociation was 150 h.

Animals↗
PubMed1983

Reassembly of lipid-protein complexes of pulmonary surfactant. Proposed mechanism of interaction.

We studied the interaction at 37 degrees C between a major apolipoprotein of pulmonary surfactant and 11 mixtures of lipids. The experiments were carried out in the presence of either 3 mM Ca2+ or 10 mM EDTA. The amount of apolipoprotein associated with lipid was independent of Ca2+. However the binding was sensitive to the percentage of gel-state lipid in the vesicles, and the amount of apolipoprotein in the recombinant lipoprotein complex decreased as the percentage of fully saturated phospholipid was reduced. Maximum association of the apolipoprotein occurred with lipid vesicles containing 85% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 15% 1,2-dipalmitoyl-sn-glycero-3-phospho-1-glycerol or 1,2-dipalmitoyl-sn-glycerol. Fluorescence measurements on the apolipoprotein indicated that the tryptophan side chains were in a relatively hydrophobic environment, and that the wavelength of maximum fluorescence emission was not changed upon the binding of lipid. The results suggest that the principal mode of interaction between the apolipoprotein and lipids of surfactant is hydrophobic bonding. The most extensive binding occurs with lamellar lipids in a gel that would be expected to have inhomogeneities in packing density due to the presence of acidic phospholipids or other glycerolipids. The role of Ca2+ in this interaction has not been fully determined. Although it is not needed to effect the binding of the lipids and the apolipoprotein, it does influence the physical state of the complex, and possibly the stoichiometry of lipid to protein. Some of the processes mediated by Ca2+ in this interaction may be analogous to those observed in membrane fusion. Thus, Ca2+ probably causes segregation of the lamellar phospholipids into domains, inducing vesicular disruption and fusion. This lipid aggregates about hydrophobic sites on the protein, thereby forming high molecular weight reassembly complexes.

Animals↗
PubMed1983

A study of the quenching of the intrinsic fluorescence of succinyl-CoA synthetase from Escherichia coli by acrylamide, iodide, and coenzyme A.

Escherichia coli succinyl-CoA synthetase (SCS) contains three tryptophan residues per mole of alpha beta dimer, and all of them are on the beta subunit. SCS shows an emission maximum at 335 nm which is shifted to 350 nm upon denaturation by urea or guanidine hydrochloride. Acrylamide is able to quench the tryptophan fluorescence in SCS by static and dynamic mechanisms. Substrates give protection against quenching by acrylamide. Binding of ATP to the alpha subunit which has no tryptophans gives as large an effect on the quenching by acrylamide as the binding of coenzyme A (CoA) to the beta subunit. Addition of CoA eliminates the curvature observed in Stern-Volmer plots for acrylamide quenching obtained by lifetime measurements. Potassium iodide does not quench the SCS fluorescence in the presence of CoA. These results suggest that there are heterogeneously emitting tryptophan residues in SCS that are located at the alpha beta subunit contact region close to the CoA binding site. Hence, the tryptophan residues can act as intrinsic reporters of events taking place at the active site of this enzyme. Further, the present results support models for SCS that put the active site at the alpha beta subunit contact region.

Acrylamide↗
PubMed1982

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.

Bacterial Proteins↗
PubMed1982

Controlled proteolysis by subtilisin as a probe for cyanide-induced conformational changes in Pseudomonas cytochrome oxidase.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c-551:oxidoreductase; EC 1.9.3.2), digested with subtilisin in the absence and presence of KCN, produces discrete, high molecular weight fragments. The presence of KCN alters the rate of this fragmentation but does not change the nature of the fragments. When digested in the absence of KCN, the oxidase gives a major product (A) which is enzymatically active and has an apparent Mr = 58,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of KCN, the major product (B) has Mr = 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but with gel permeation high performance liquid chromatography it has an apparent Mr = 92,000. This implies that product B is dimeric, as is the parent enzyme which has Mr = 110,000 by high performance liquid chromatography. Absorption spectra of product B, isolated by gel filtration, show that it contains only the heme d1 moiety. The digestion time course indicates that the rate at which several minor products are formed is also dependent on the absence or presence of KCN. These observations suggest that the binding of KCN to the heme centers induces a conformational change in the enzyme so that the heme c-containing portion of the protein, which is at one end of the intact enzyme, can be removed without disrupting the integrity of the heme d1-containing portion.

Chromatography, High Pressure Liquid↗
PubMed1982

A fluorescence study of conformational changes induced by substrate and temperature in bovine liver thiosulfate sulfurtransferase.

Structural transitions occurring in the range of 0-50 degrees C have been detected and studied in the enzyme thiosulfate sulfurtransferase (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) by investigating both the intrinsic protein fluorescence and the fluorescence of covalently bound probes. The intrinsic fluorescence of the enzyme decreases sharply at 27 degrees C and the magnitude of this quenching is smaller for the sulfur-substituted enzyme (ES) than for the free enzyme (E), both of which are obligatory catalytic intermediates. The effect with ES is fully reversible and almost completely so with E. Fluorescence depolarization sudies with thiosulfate sulfurtransferase labeled at a number of different sites with the fluorosphore dimethylaminonaphthalene show a sharp increase in the polarization starting at 27 degrees C when the temperature/viscosity ratio is varied with temperature and no transition when the ratio is varied with glycerol. Enzyme activity show no transition at 20 degrees C but falls abruptly above 40 degrees C. Under the appropriate conditions, the 27 degrees C transition will lead to association of thiosulfate sulfurtransferase molecules and the appearance of turbidity. Both activity and fluorescence measurements support the idea that the ES form is significantly more stable than the E form. These results may result from changes in the interactions between the structural domains into which the single polypeptide chain of thiosulfate sulfurtransferase is folded.

Animals↗
PubMed1976

The aromatic residue content of the enzyme rhodanese.

The aromatic amino acid composition of the enzyme rhodanese has been redetermined. Previous reports have varied from 5 to 11 tryptophans per 26 alanine residues. The present work has quantitated the aromatic residues by a combination of amino acid analysis, solvent perturbation difference spectroscopy, specific residue modification and direct ultraviolet spectral analysis. These methods indicate that rhodanese contains 10 tyrosines, eight tryptophans and 16 phenylalanines per 26 alanine residues. The results for tyrosine and phenylalanine are in reasonable agreement with previous results.

Animals↗
PubMed1976

Spectral studies of the tryptophan exposure in the enzyme rhodanese.

Tryptophan exposure in the enzyme rhodanese (thiosulfate:cyanide sulfur-transferase, EC 2.8.1.1.) has been studied by the methods of solvent perturbation difference spectroscopy, fluorescence spectroscopy and fluorescence quenching. The data from all these techniques are consistent with two classes of tryptophan:surface and buried. The surface residues appear to be relatively shielded and in an anionic environment. The buried residues have spectral characteristics suggesting they are in a very apolar environment and have less than the expected contact with the peptide backbone. The solvent perturbation difference spectra indicate further that additional tyrosine as well as tryptophan residues are buried as rhodanese is converted from the free enzyme to the sulfur-substituted enzyme: forms which are obligatory intermediates in the catalytic cycle.

Animals↗
PubMed1976

Denaturation-induced disulfide formation in the enzyme rhodanese.

The effect of denaturants on the quantitation of free sulfhydryl groups in the enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) has been reinvestigated in some detail. The sulfhydryl assay with the colorimetric reagent 5, 5'-dithio-bis (2-nitrobenzoic acid) Nbs2 shows four sulfhydryl groups per enzyme molecule (mol. wt. 32 300) when the colorimetric reagent is added to the assay mixture before the denaturant, sodium dodecyl sulfate. On the other hand, only two sulfhydryl groups per molecule are observed when Nbs2 is added after denaturation has been initiated. The time dependence observed in this latter procedure indicates that the loss of the two groups is rapid and permanent. The results depend on the denaturant used: urea acts like sodium dodecyl sulfate while guanidine reveals four sulfhydryl groups independent of reagent order. The assay also gives four sulfhydryl groups independent of reagent order. The assay also gives four sulfhydryl groups independent of reagent order with urea or sodium dodecyl sulfate under conditions which are expected to limit metal ion-catalyzed oxidation of sulfhydryl groups (e.g. oxygen exclusion or metal ion chelation). Recent studies have shown that rhodanese has a molecular weight of 32 600, no disulfides and four sulfhydryl groups per molecule. These results together with the observations reported here are taken to indicate that a disulfide can be formed during denaturation of rhodanese and that the pathway of denaturation determines the result obtained.

Binding Sites↗