Bernard Axelrod

Isolation of an isozyme of soybean lipoxygenase

Abstract 1. 1. An isozyme, distinct from the well-known crystalline lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.1.13, formerly known as lipoxidase) of Theorell, has been isolated from soybeans, as an electrophoretically homogeneous protein. 2. 2. Its substrate specificity over a range of pH values indicates that the newly purified enzyme may be responsible for the “triglyceride” lipoxidase activity reported by Koch. 3. 3. The new enzyme is distinct from the originally described lipoxidase of Theorell by the following criteria: elution profile from DEAE-Sephadex, disc gel electrophoresis, pH activity profile, specificity and heat stability.

Isolation of a third isoenzyme of soybean lipoxygenase

Abstract 1. 1.|A third isoenzyme of soybean lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.1.13) has been purified to essential homogeneity by Sephadex DEAE-A50 column chromatography and isoelectric focusing. 2. 2.|The enzyme differs from the two previously described pure isoenzymes (lipoxygenases-1 and -2)1 by the following criteria: elution profile from DEAE-Sephadex, isoelectric point, pH activity profile, and the effect of Ca2+ on activity. 3. 3.|The new isoenzyme also shows an anomalous, inverse dependence of activity on enzyme concentration.

Double dioxygenation of arachidonic acid by soybean lipoxygenase-1

Abstract Soybean lipoxygenase-1, has been found to catalyze the incorporation of two oxygen molecules into arachidonic acid. The product appears to be 8,15 dihydroperoxy-5,9,11,13-eicosatetraenoic acid. This is apparently the first report of the enzymatic production of a conjugated aliphatic triene in vitro .

1-Aminoglycosides, a new class of specific inhibitors of glycosidases☆

Abstract 1-Aminoglycosides represent a new class of specific and relatively potent inhibitors of glycosidases. These compounds are specific against those enzymes which act upon glycosides that correspond to glycone of the inhibitor. Thus α- and β- D = - are inhibited by D = -glucosidases but not by D = -galactosylamine and D = -mannosylamine. α- and D = -galactosidases are inhibited by D = -galactosylamine but not by the other two glycosylamines. D = -Mannosylamine inhibits mannosidase.

Evidence for a single catalytic site on the “β3-d-glucosidase-β-d-galactosidase” of almond emulsin☆

Abstract An isoenzyme of glycosidase obtained from almond emulsin, which is both a β- d -glucosidase and a β- d -galactosidase, has now been shown to possess β-D-fucosidase activity. It has been concluded that all three activities reside in a single catalytic site for the following reasons. (i) d -Glucosylamine, d -galactosylamine, and d -fucosylamine (a newly discovered potent inhibitor of this enzyme) each act competitively against all three of the substrates. (ii) Any given inhibitor exhibits the same K i value when tested in the presence of any of the three substrates, (iii) When the enzyme is incubated with any two of the p -nitrophenyl glycoside substrates, at or above their respective K m values, the rate of p -nitrophenol formation is not additive, but rather is equal to the value calculated on the basis of the individual K m values and relative maximum velocities.

Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli☆

We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.

Factors influencing the positional specificity of soybean lipoxygenase

1. 1. The positional specificity of hydroperoxidation of two isoenzymes (lipoxygenases-1 and -2) of the lipoxygenase (linoleate: O2 oxidoreductase, EC 1.13.1.13) from soybean has been investigated under a number of experimental conditions, i.e. temperature, substrate, pH, O2 tension, and presence or absence of Ca2+. 2. 2. Values for the ratio of 9- and 13-hydroperoxide isomers were calculated after combined gas-liquid chromatographic-mass spectral analysis of the corresponding 9- and 13-hydroxymethyl stearates derived from the hydroperoxides by chemical modification. 3. 3. The results show that not only are the experimental conditions under which the enzyme is incubated important in determining the position oxygenated but also the composition of the lipoxygenase preparation with regard to its isoenzyme content. 4. 4. Results obtained with methyl linoleate as substrate indicate that the variation in values for isomer ratios is not unique to the linoleic acid substrate.

On the different positional specificities of peroxidation of linoleate shown by two isozymes of soybean lipoxygenase.

Summary A number of contradictory reports concerning the products of hydroperoxidation of linoleate as catalyzed by soybean lipoxygenase have appeared with claims ranging from 100% 13-hydroperoxyoctadecadienoate to 70% 13-hydroperoxyoctadecadienoate: 30% 9-hydroperoxyoctadecadienoate. Determination of the specificity of two homogeneous isozymes of soybean lipoxygenase offers a resolution of this anomaly. The first of these enzymes, the “classical” lipoxygenase originally crystallized and described by Theorell et al . (1) (lipoxygenase-1 of Christopher et al . (2)) yields only the 13-isomer. The second isozyme, lipoxygenase-2 (2), forms a 50:50 mixture of the 9- and 13-isomers.

Multiple dioxygenation by lipoxygenase of lipids containing all-cis-1, 4, 7-octatriene moieties.

Abstract Soybean lipoxygenase-1 acting upon polyunsaturated fatty acids containing a suitably positioned all- cis -1,4,7-octatriene moiety generates bishydroperoxy derivatives while consuming approximately 2 mol of O 2 per mole of substrate. The reaction has been separated into two steps: The first is marked by the rapid monohydroperoxidation of the starting material and the second is marked by the much slower hydroperoxidation of the first product. All of the compounds which are successfully converted to bishydroperoxy derivatives contain an ω6,9,12 grouping of cis double bonds. α-Linolenic acid (ω3,6,9) cannot serve as a substrate because its preferred site of oxygenation is in the center of the octatriene moiety. The K m for arachidonic acid in the reaction leading to the singly hydroperoxidized monohydroperoxide is 8.6× 10 −5 , m , which is approximately 200 times smaller than the K m for the monohydroperoxide in the second step leading to the bishydroperoxide. All of the substrates which undergo double dioxygenation form conjugated trienes.

Effect of substrate polarity on the activity of soybean lipoxygenase isoenzymes

In order to characterize the several isoenzymes of soybeans, they were examined with respect to the effect of the polar nature of the substrate. In general, lipoxygenase-1 was most active when presented with charged substrates such as the anionic form of linoleic acid or of potassium linoleyl sulfate, whereas lipoxygenase-2 and-3 preferred nonpolar substrates such as unionized linoleic acid, methyl linoleate, linoleyl methane sulfonate, 10,13-nonadecadieneamine, or linoleyl acetate. Linoleyl sulfate, which has been advanced as an excellent readily soluble substrate for lipoxygenase, was indeed the best substrate found for lipoxygenase-1. Lipoxygenase-2 and-3 were, by contrast, totally inactive against this substrate. The favorable response of linnoleic acid to lipoxygenase-2 and-3 at pH 6.8 was ascribed to the anomalously high pKa value of linoleic acid compared to that of short chain carboxylic acids. The pH-activity profile obtained with lipoxygenase acting on linoleyl sulfate (which was charged at all pH values examined) was shifted to lower pH values compared to the linoleic acid activity profile. The effect of changing from the charged to the uncharged substrate, when tested against lipoxygenase-1, was to increase the Km by an order of magnitude.