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W W Fish

Publications and source records attributed to W W Fish.

At least 37 records · Page 2Linked to original sources
PubMed1982

On the reliability of the use of heparin immobilized on agarose for the study of the interactions among heparin, thrombin and antithrombin.

The extent of inhibition of thrombin was re-examined as a consequence of the sequence of addition of thrombin and antithrombin III to a column of heparin immobilized on agarose. With the use of pure enzyme, pure inhibitor, and a highly sensitive chromogenic substrate, no appreciable difference in the extent of thrombin inhibition was observed between the two sequences of addition. These observations, together with a demonstrated sensitivity of the method to variations in experimental protocol, challenge the conclusions reached in an earlier work (Hatton and Regoeczi, Thromb. Res. 1977; 10:645) which utilized this experimental approach but which employed larger quantities of reactants and a less sensitive substrate.

Animals↗
PubMed1982

Purification and macromolecular properties of a sialic acid-specific lectin from the slug Limax flavus.

A lectin (LFA) which is highly specific for sialic acid has been purified from the slug Limax flavus by a combination of ammonium sulfate fractionation and affinity chromatography on bovine submaxillary mucin coupled to Sepharose 4B. The affinity-purified lectin appeared homogeneous by electrophoresis in the presence of sodium dodecyl sulfate. Below 1 mg/ml at pH 7, LFA exists as a species of Mr = 44,000 which is composed of two equal sized subunits. Above 1 mg/ml, the protein solution was observed to behave as a rapidly associating-dissociating system. N-acetylneuraminic acid and N-glycolylneuraminic acid gave a 50% inhibition of agglutination of erythrocytes by LFA at 0.13 and 0.81 mM, respectively. Galactose, N-acetylgalactosamine, galactosamine, glucose, N-acetylglucosamine, glucosamine, mannose, arabinose, xylose, fucose, glucuronic acid, alpha-methyl-D-glucoside, alpha-methyl-D-mannoside, lactose, and sucrose were ineffective inhibitors at concentrations up to 10-25 mM. Bovine submaxillary mucin, a sialoprotein, was a potent inhibitor of hemagglutination by LFA. Upon treatment of the mucin with neuraminidase, loss of inhibitory activity was observed which was proportional to the loss of sialic acid from the mucin.

Amino Acids↗
PubMed1981

A re-examination of some properties of fatty acyl-CoA micelles.

Three separate techniques have been employed to estimate the critical micelle concentration: spin labeling using 6-doxylstearoyl-CoA, gel permeation chromatography, and analytical ultracentrifugation. The first method is a labeling technique. The latter two methods utilize no potentially interfering probe and provide a value for the aggregation number for palmitoyl-CoA. All three methods provide a critical micelle concentration for palmitoyl-CoA no lower than 30 to 60 microM. The latter methods provide an aggregation number near 40 and certainly no larger than 200. These values are inconsistent with the values suggested earlier (Zahler, W. L., Barden, R. E., and Cleland, W. W. (1968) Biochim. Biophys. Acta 164, 1-11). The spin-labeled analogues, 6- and 16-doxylstearoyl-CoA, were shown not to micellize, yet these analogues were good inhibitors for citrate synthase. These observations will require the re-examination of a large body of literature in which inhibition of enzymes by fatty acyl-CoA at concentrations below 30 microM was simply ascribed to the formation of micelles.

Acyl Coenzyme A↗
PubMed1980

Behavior of glycopolypeptides with empirical molecular weight estimation methods. 1. In sodium dodecyl sulfate.

The influence of the presence of oligosaccharide branches was examined with respect to the behavior of glycopolypeptides in empirical molecular weight estimation methods in the presence of sodium dodecyl sulfate (NaDodSO4). This examination was conducted by comparing the gel chromatographic and gel electrophoretic behaviors in the presence of NaDodSO4 of 13 glycopolypeptides of known chemical and physical properties to those of regular polypeptides. Errors in the gel chromatographic molecular weight for glycopolypeptides in NaDodSO4 varied from -22% to +10% and indicated that the hydrodynamic behavior of the glycopolypeptide--NaDodSO4 complex could not be correlated with the amount of carbohydrate in the glycopolypeptide. NaDodSO4 binding measurements on a number of the glycopolypeptides suggest that the polypeptide moiety binds the nominal weight ratio of NaDodSO4, while the carbohydrate portion exhibits little or no NaDodSO4 binding. As has been reported by others, the polyacrylamide gel electrophoretic behavior of glycopolypeptide--NaDodSO4 complexes yielded abnormally high molecular weight estimates. In general, the error of these estimates diminished with decreasing porosity of the gel; however, each glycopolypeptide behaved in a unique fashion. Treatment of the electrophoretic data by any of several empirical means provided no reliable way to correct for the glycopolypeptides' aberrant behavior.

Chromatography, Gel↗
PubMed1980

Behavior of glycopolypeptides with empirical molecular weight estimation methods. 2. In random coil producing solvents.

The effects of oligosaccharide branch chains on the hydrodynamic behavior of reduced glycopolypeptides was examined by gel chromatography in random coil producing solvents. This entailed a comparison of the gel chromatographic behavior in the presence of concentrated guanidinium chloride of 16 glycopolypeptides of known physical and chemical properties to that of regular polypeptides. For most of the glycopolypeptides employed, the presence of oligosaccharide branches sufficiently perturbed the dimensions of the unfolded glycopolypeptide such that its effective hydrodynamic radius was the same as that of a linear polypeptide of the same total mass. For this reason, gel chromatography in random coil producing solvents appears to be the most reliable empirical method to obtain a first approximation of the molecular weight of a glycopolypeptide. Glycopolypeptides rich in N-acetylneuraminic acid, and thus possessing low isoionic points, exhibited more pronounced deviations in their electrophoretic behavior in the presence of 8 M urea than those glycopolypeptides whose ionic properties were similar to those of the polypeptide standards employed.

Carbohydrate Conformation↗
PubMed1979

Routes of thrombin action in the production of proteolytically modified, secondary forms of antithrombin-thrombin complex.

The reaction between thrombin and antithrombin results in the formation of an inactive, stable, equimolar complex between the two proteins. However, under most reaction conditions several secondary complex forms, which have lower apparent molecular weights in dodecyl sulfate/polyacrylamide gel electrophoresis, appear concomitantly with or immediately following the production of the primary form of the complex. Purification of nascent, intact complex and treatment of this complex form with thrombin demonstrated that these subsidiary forms of antithrombin-thrombin complex may arise by proteolysis of the nascent complex by excess thrombin. Dissociation of such proteolytically modified complex preparations by hydroxylamine, and examination of the dissociation products by dodecyl sulfate/polyacrylamide gel electrophoresis suggested that degradation occurs primarily in the thrombin part of the complex, and only after prolonged proteolysis in its antithrombin moiety also. Incubation of antithrombin with several autolytically modified thrombin preparations showed that formation of subsidiary complex forms can also occur by an alternative route, i.e. between premodified thrombin forms and the inhibitor. In contrast, complex formation between thrombin and active forms of antithrombin, which have been modified by thrombin before complex formation, is unlikely, since no such active forms of antithrombin could be demonstrated.

Animals↗
PubMed1977

The properties of Corbicula sandai apoferritin.

Corbicula sandai apoferritin possesses physical properties different from apoferritins of other species. The native molecular weight was estimated from its s020,w of 18.7 S to be about 503 000. Empirical molecular weight estimation methods in denaturing solvents yielded a molecular weight estimate for the constituent polypeptide chain of 23 000. The circular dichroic spectrum of C. sandai apoferritin was significantly different from other apoferritins and it was immunologically unreactive with rabbit anti-human ferritin antisera.

Amino Acids↗
PubMed1977

The subunit characterization of Callinectes sapidus hemocyanin.

Hemocyanin from the blue crab, Callinectes sapidus, sediments at 25.7 S and has a native molecular weight of 940 000 +/- 20 000. Under solution conditions of increased pH (approximately 10) or ionic strength, the native molecule dissociates to a 17 S species. Reversal of this dissociation was unsuccessful. At pH 10 and with the removal of Mg2+, the 17 S species reversibly dissociates to form a subunit species which sediments at 6 S. A comparison of the circular dichroic spectra of the 25.7 S and 6 S hemocyanins suggests that little happens to the structural integrity of the polypeptide backbone upon the two dissociations. Molecular weight estimations under reducing and denaturing conditions indicate that the 6 S hemocyanin species represents the constituent polypeptide chain of the protein molecule. Chemical analysis suggests the presence of a small amount, less than 3%, of carbohydrate bound to the polypeptide chain. Electrophoresis of the hemocyanin in the presence of sodium dodecyl sulfate or urea reveals two major electrophoretic species of either slightly different chemical composition or slightly different polypeptide chain length.

Animals↗
PubMed1977

Isolation and characterization of rat alpha-lactalbumin: a glycoprotein.

alpha-Lactalbumin was purified to homogeneity from rat milk. Rat alpha-lactalbumin, in contrast to other alpha-lactalbumins, is a glycoprotein and exhibits an abnormally high molecular weight when obtained by gel filtration or electrophoresis in sodium dodecyl sulfate. The molecular weight by sedimentation equilibrium is 15 400 +/- 5% and of the reduced and alkylated protein is 16 000 when determined by thin-layer chromatography in 6 M guanidine hydrochloride. At least, three major charge forms, all containing carbohydrate and active in the lactose synthetase reaction were demonstrated. The amino acid composition reveals a high proline content which is reflected in a low alpha-helical content.

Amino Acids↗
PubMed1977

Physical properties of Tamm-Horsfall glycoprotein and its glycopolypeptide.

The molecular weight of the constituent glycopolypeptide chain of T-H glycoprotein was determined by sedimentation equilibrium under two entirely different sets of denaturing conditions. For both sets of denaturing conditions, the average molecular weight estimated for T-H glycopolypeptide was 74,000. The gel chromatographic behavior in 6M guanidium chloride of T-H glycopolypeptide with disulfide bonds intact as compared with its gel chromatographic behavior with disulfide bonds broken indicated that the glycopolypeptide is highly constrained by intrachain disulfide bonds.

Chromatography, Gel↗