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Biomedical subjects

J R Whitaker

Publications and source records attributed to J R Whitaker.

At least 55 records · Page 3Linked to original sources
PubMed1971

Purification and Properties of Chlorophyllase from Ailanthus altissima (Tree-of-Heaven).

Chlorophyllase from Ailanthus altissima leaves has been purified 63-fold by a combination of heat treatment, ultracentrifugation, gel filtration, and chromatography on diethylaminoethyl cellulose. While the enzyme is inhibited to some degree by Triton X-100, a modification of the assay procedure of Klein and Vishniac has been shown to be far superior to the use of aqueous acetone systems. The enzyme was found to have a pH optimum on pheophytin a of 4.5. Chlorophylls a and b, pheophytins a and b, and pyropheophytin a were hydrolyzed by the enzyme while protochlorophyll a and 4-vinyl protochlorophyll a were not hydrolyzed but were competitive inhibitors. p-Nitrophenyl acetate was not hydrolyzed. The enzyme does not appear to contain an essential sulfhydryl group since sodium tetrathionate and p-chloromercuribenzoate did not affect its activity.

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PubMed1971

Isolation and characterization of polyphenol oxidase isozymes of clingstone peach.

The polyphenol oxidase system in clingstone peach (Prunus persica) was investigated. Polyacrylamide disc-gel electrophoresis indicated four bands with polyphenol oxidase activity in extracts from acetone powder of clingstone peach. These four isozymes were then isolated from a buffer extract of peach acetone powder by cold acetone precipitation, followed by diethylaminoethyl cellulose column chromatography. All isozymes had different heat stabilities. At 55 C, polyphenol oxidases A, B, and D had half-lives of 5.4, 14.6, and 14.1 minutes, respectively. Polyphenol oxidase C was stable over a period of 50 minutes of incubation at 55 C, but had a half-life of 2.2 minutes at 76 C. None of the isozymes had monophenolase activity, and they varied in their specificity for several diphenols. The following values were found for polyphenol oxidases A, B, C, and D, respectively, with catechol as substrate: optimal pH: 6.8, 6.5, 7.2, and 7.0; Michaelis constant: 6.6, 4.2, 7.0, and 36 mm; V(max)/(E(0)): 4.95, 39.4, 2.16, and 80.0 (DeltaA min(-1) mg(-1)). Each isozyme showed a different amount of inhibition by NaHSO(3), NaCl, NaCN, l-ascorbic acid, glutathione, ethylenediaminetetraacetate, and sodium diethyldithiocarbamate.

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PubMed1971

Effect of Phloroglucinol and Resorcinol on the Clingstone Peach Polyphenol Oxidase-catalyzed Oxidation of 4-Methylcatechol.

Phloroglucinol and resorcinol are not substrates for clingstone peach (Prunus persica) polyphenol oxidase, but they react with 4-methyl-o-quinone, produced either enzymatically or nonenzymatically, to give an intense red or red-brown color with a maximal absorption at about 470 nanometers. Several colored products were isolated from an ethyl acetate extract of the reaction by two-dimensional thin layer chromatography. Based on thin layer chromatographic and spectral studies of the enzymatic and nonenzymatic reactions, polyphenol oxidase does not play a role in the reaction between 4-methyl-o-quinone and phloroglucinol, resorcinol, d-catechin, or orcinol. In such reactions, the function of polyphenol oxidase is the formation of 4-methyl-o-quinone which then reacts nonenzymatically with the above phenols. Activation energies of both enzymatic and nonenzymatic reactions were determined.

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PubMed1969

Multiple molecular forms of ficin-evidence against autolysis as explanation.

While the total amount of proteolytic enzyme activity in the latex from Ficus carica varieties Kadota and Black Mission showed seasonal variation and was different between the 2 crops of figs in the latex collected at the same time and from the same tree, there is no difference in the number of multiple molecular forms of ficin present as demonstrated by chromatography on carboxymethyl-cellulose. The number of multiple forms of ficin is also the same, with 1 exception, among samples of latex which have been collected with no protection against proteolysis, samples which have been collected directly into sodium p-chloromercuribenzoate to inhibit all proteolysis and samples which have been held at an elevated temperature so as to encourage proteolysis. Evidence is presented to support the proposal that component C of Ficus carica variety Kadota is not present in the original latex but that it is produced from component D during the purification procedure. The data support the hypothesis that, with the possible exception of 1 component, the multiple molecular forms of ficin are not the result of artifacts produced by autolysis during collection, storage and purification of the sample.

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PubMed1969

Nature of the Conversion of Ficus carica Variety Kadota Ficin Component D to Component C. Some Physicochemical Properties of Components C and D.

Component C can be formed from component D under the experimental conditions used during purification of Ficus carica variety Kadota latex. By use of the inhibitor, sodium p-chloromercuribenzoate, the 2 components have been purified to chromatographic homogeneity. The 2 components have identical molecular weights and amino acid composition. The only difference found between the 2 components is the presence of 3 to 6 more amide groups in component D than in component C. There also appears to be a conformational difference between the 2 since component C is not as acidic, with respect to component D, as would be expected from the comparative amide contents. Conformational differences between the 2 are also indicated by the chromatographic behavior of the 2 in the presence and absence of sodium-p-chloromercuribenzoate.

Journal Article↗
PubMed1969

Multiple Molecular Forms of Ficus glabrata Ficin. Their Separation and Relative Physical, Chemical, and Enzymatic Properties.

Six of the proteolytic enzyme components of Ficus glabrata ficin have been isolated and shown to be chromatographically homogeneous. The molecular weights, the amino acid compositions, the electrophoretic and chromatographic behavior of the tryptic peptides, and the relative specificities of these 6 components have been determined. Within the experimental precision of the methods all 6 components are identical. They also have identical solubilities in sodium chloride and ammonium sulfate solutions. However, they are markedly different in their chromatographic properties. These multiple molecular forms of Ficus glabrata ficin may differ only in their conformational forms (conformers) or they may have minor differences in amino acid sequences which are sufficient to give different conformations and yet not be detected by the usual peptide mapping techniques. At the moment, we favor the latter possibility.

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PubMed1969

Ficin-Catalyzed Reactions. Hydrolysis of alpha-N-Benzoyl-l-Arginine Ethyl Ester and alpha-N-Benzoyl-l-Argininamide.

The effect of pH on the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and alpha-N-benzoyl-l-argininamide (BAA) by a proteolytic enzyme component purified from Ficus carica var. Kadota latex has been studied in detail over the pH range of 3 to 9.5. k(cat) (lim) values for the hydrolysis of BAEE and BAA were essentially identical (5.20 and 5.01 sec(-1), respectively at 30 degrees ). k(cat) values for hydrolysis of BAEE and BAA were dependent on prototropic groups with apparent pK values of 4.24 and 8.53 and 4.10 and 8.59, respectively. k(cat) (lim) values for tht hydrolysis of BAEE and BAA were essentially identical (5.20 and groups of pK 4.33 and 8.60 and 4.55 and 8.51, respectively. Thus the pH optimum is 6.5 for both substrates. K(m) (app) values for BAEE and BAA were 3.32 x 10(-2)m and 6.03 x 10(-2)m respectively over the pH range of 3.9 to 8.0. These data are interpreted in terms of the involvement of a carboxyl and a sulfhydryl group in the active center of the enzyme. The data do not support the concept that deacylation of the acyl-enzyme is completely the rate controlling step in the hydrolyses. Rather, it appears that the magnitude of k(2) and k(3) are not greatly different.

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PubMed1968

Proteolytic activity in the genus ficus.

The latices of only 13 of a total of 46 species of Ficus examined contained appreciable proteolytic activity. Therefore, high proteolytic activity in the latex is not a distinguishing feature of the genus. The latex of F. stenocarpa had the highest specific activity followed closely by the latices of F. carica and F. glabrata. Latices of 6 species of Ficus were examined by chromatography on CM-cellulose and compared with the results obtained for 9 varieties of F. carica. All of the latices were found to contain multiple proteolytic enzymes. Chromatographically, the multiple enzyme components of the several varieties of F. carica were more similar than those of the several species examined. The latices of 16 varieties of F. carica were all different as determined by free boundary electrophoresis although the specific proteolytic activity of the latices was reasonably constant.

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