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Biomedical subjects

B Axelrod

Publications and source records attributed to B Axelrod.

At least 37 records · Page 2Linked to original sources
PubMed1983

Identification of the target amino acids in the site-specific inactivation of triose phosphate isomerase by ferrate anion.

Ferrate anion, an analog of orthophosphate anion, very rapidly inactivates triose phosphate isomerase from chicken muscle. The inactivation can be prevented by the presence of competitive inhibitors. Of the 247 amino acids known to be present in each of the identical monomers of this dimeric enzyme, Trp-168, located in the active site pocket, as well as Trp-191 and His-248 are destroyed. The partial loss of Tyr-164 also occurs. Trp-168 is known from published crystallographic studies to be located in the active site cavity harboring Glu-165. The gamma-carboxylate group of Glu-165 is believed to serve as the nucleophile which catalyzes the isomerization. Tyr-164, Glu-165, and Trp-168 are known to be conserved in all of the triose phosphate isomerases which have been sequenced, including those obtained from mammals, chicken, fish, yeast, and bacteria. It is suggested that the chemical modification of Trp-168 alters its shape and hydrophobic character in a manner that adversely affects the conformation of the active site cavity. When the chicken enzyme is treated with ferrate in the presence of the competitive inhibitor phosphoglycolate, only His-248 is destroyed. Thus, His-248, which is the COOH-terminal amino acid, cannot be essential for activity. This observation is consistent with the knowledge that it is not invariant in the enzyme from various species.

Amino Acids↗
PubMed1983

Effect of albumin on binding and recovery of enzymes in affinity chromatography on Cibacron Blue.

Bovine serum albumin appears to improve the specificity of Cibacron Blue F3GA in affinity chromatography of enzymes which interact with nucleotides. The action of bovine serum albumin may rest in its ability to selectively mask affinity sites in the dye, which are not specific for the nucleotide-binding region of the enzyme, while not seriously impairing binding nor its elution by nucleotides. Thus, the elution of Chlorella nitrate reductase from a Blue Sepharose chromatographic column by its coenzyme, NADH, fails, unless the column is first treated with bovine serum albumin. Such treatment also improves the recovery of some other nucleotide-binding enzymes tested.

Anthracenes↗
PubMed1979

Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion.

Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.

Amino Acids↗
PubMed1978

Biosynthesis of a prostaglandin by a plant enzyme.

Lipoxygenase-2 from soybeans catalyzes the oxygenation of arachidonic acid to form significant amounts of a prostaglandin product. Results obtained with the dithionite-reduced derivatized product upon combined gas chromatography-mass spectroscopy are consistent with structures of the stereoisomers of 9,11,15-trihydroxyprosta-5,13-dienoic acid. A portion of the material formed by reduction of the enzyme product with dithionite reacts positively in a radioimmunoassay against rabbit anti-prostaglandin F2alpha antibody. This appears to be the first report of the synthesis of a prostaglandin by a non-animal enzyme.

Arachidonic Acids↗
PubMed1977

Characterization of two isoenzymes of lipoxygenase from bush beans.

Two isoenzymes of lipoxygenase, a and b, have been obtained from bush beans (Phaseolus vulgaris) as electrophoretically homogeneous proteins. Both proteins have a molecular weight of 100,000, contain 1 atom of iron, and appear to be composed of a single peptide chain. However, these enzymes appear to differ in some other respects. Thus, lipoxygenase a has an isoelectric point of 6.03 while lipoxygenase b has a value of 5.57. Their pH optima are 5 to 7 and 6.5 to 7, respectively. Both lipoxygenase a and b, when acting on linoleic acid plus the product hydroperoxide, generate what are presumably keto-dienes with an absorption maximum at 280 nm. Whereas lipoxygenase a can catalyze this secondary reaction in the presence of O(2), lipoxygenase b does so only under anaerobic conditions. Lipoxygenase a is stimulated by Ca(2+) while lipoxygenase is not. An unexpected finding is the strong inhibition of lipoxygenase a by Mn(2+) (50% inhibition at 12.5 muM under standard reaction conditions). Lipoxygenase b is inhibited by Mn(2+) but only at concentrations about 250 times greater.

Journal Article↗
PubMed1976

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).

Electron Spin Resonance Spectroscopy↗
PubMed1975

The specificity of the synthetic reaction of two yeast alpha-glucosidases.

The specificity of the hydrolytic reaction has been compared to that of the synthetic reaction for maltase and isomaltase (alpha-methyl-D-glucosidase) from Saccharomyces oviformis. Maltase which hydrolyzes the alpha-1,4-disaccharide, maltose, and the alpha-1,6-disaccharide, isomaltose, catalyzes the formation of both maltose and isomaltose from free glucose. Isomaltase, which hydrolyzes isomaltose but not maltose, catalyzes the formation only of isomaltose from glucose. Both enzymes hydrolyze p-nitrophenyl-alpha-D-glucoside releasing the alpha-anomer of glucose. The enzymes utilize the alpha-anomer but not the beta-anomer for the synthesis of the disaccharides. These results are consistent with the double displacement mechanism for glycosidases and with the proposal that the glucosyl-enzyme complex is an intermediate in the reaction. The competitive inhibition by D-glucose is independent of its anomeric form for both enzymes.

Borohydrides↗