Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.     

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.     

Pyrrolooxygenase isoenzymes from wheat germ

Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.     

Further characterization of serum alkaline phosphatase from male and female beagle dogs

Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.     

Isoenzyme composition and some properties of pea lipoxygenase]

The isoenzyme composition and some properties of lipoxygenase isolated from the seeds and 10-day old sprouts of pea plant were studied. The enzyme activity assay, using gel- and ion-exchange chromatography, disc-electrophoresis in polyacrylamide gel, etc. revealed that the plant contains two lipoxygenase systems of unsaturated long-chain fatty acids oxidation. The existence of four lipoxygenase isoenzymes whose combination determines the type of lipoxygenase-catalyzed reactions of linoleic acid oxidation, has been confirmed.     

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

The differential effect of disulfiram on lipoxygenases from Glycine max

A kinetic analysis of the effect of disulfiram on the isoenzymes of lipoxygenase from soybean has been carried out. The compound is an effective inhibitor of type-2 isoenzymes but has no effect on the type-1 isoenzyme under the conditions employed in this study. The inhibitory effect is reversible and therefore does not result from covalent modification of the enzyme. The inhibition is manifest as a prolongation of the lag phase commonly seen in progress curves for lipoxygenases rather than as a reduction of the catalysed rate. A variety of structurally related and unrelated compounds have been investigated to identify the nature of the inhibitory effect. The antioxidant properties of disulfiram account for its ability to inhibit type-2 lipoxygenases. The inhibitory effect of antioxidants on type-2 lipoxygenase is only partly reversed when product hydroperoxide is included in the incubation mixture. These observations support the conclusion that free radical intermediates are integral to the catalytic mechanism of type-2 lipoxygenases.     

The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate

Summary Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.This study was supported by a grant from the Prinses Beatrix Fonds, s'Gravenhage     

Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves

The chloroplast and cytoplasmic isoenzymes of phosphoglycerate kinase (PGK) (EC. 2.7.2.3) from Hordeum vulgare leaves have been separated and purified for the first time to apparent homogeneity. The method for purifying the isoenzymes is described here and consists of DEAE Sephacel chromatography followed by affinity chromatography on ATP Sepharose. This consistently provided a 500- to 900-fold purification of each isoenzyme. Most of the total PGK in green barley leaves was found to be in the chloroplasts with only 10% in the cytoplasm. The immunological properties of the two isoenzymes were compared. The antisera raised to the separate isoenzymes showed cross-reactivity, although there is evidence that each isoenzyme possesses some distinct epitopes. The isoenzymes differ in overall charge with isoelectric points at 5.2 and 5.4 for the chloroplast and cytoplasmic isoenzymes, respectively. Molecular mass estimations by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis provided similar values of approximately 38 kilodaltons for each isoenzyme, some 4 to 5 kilodaltons less than the values calculated from the cDNA sequences of the wheat isoenzymes. The isoenzymes have broadly similar pH optima of pH 7 to 8. The cytoplasmic isoenzyme is more thermally stable than the chloroplast isoenzyme. Further studies are now in progress to compare both the regulatory properties of the isoenzymes and also their three-dimensional structures as compared with the yeast enzyme.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.     

Preparation and characterization of monoclonal antibodies against soybean seed lipoxygenase isoenzymes

Sets of monoclonal antibodies have been prepared using two soybean seed lipoxygenase isoenzymes as the antigens. The antibodies were characterized by ELISA, Western blot analysis, immunoprecipitation, and in kinetic assays. Several antibodies displaying selectivity for the two closely related polypeptides were obtained, while the majority of the antibodies generated were crossreactive. Antibodies specific to the native and denatured forms of the two proteins were also obtained. Two of the monospecific antibodies were shown to immunoprecipitate the appropriate isoenzyme selectively from a mixture. When these antibodies were immobilized on agarose, they were successful in the immunoaffinity purification of the individual isoenzymes. In kinetic experiments certain antibodies were found to influence catalysis upon incubation with lipoxygenase. Antibodies which both inhibited and stimulated catalysis were identified.     

Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes

Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.     

Polyacrylamide gel electrophoresis of rat brain acetylcholinesterase: isoenzymes of normal rat brain

Abstract— Triton-solubilized acetylcholinesterase (EC 3.1.1.7) of rat brain was submitted to vertical flatbed polyacrylamide gel electrophoresis. Three anodally migrating isoenzyme zones with low relative mobilities could be resolved, each of which on quantitative densitometry appeared to consist of more than one subzone. More than 50 per cent of the total AChE activity was exhibited by the isoenzyme zone closest to the origin (isoenzyme zone 3). Regional differences in AChE isoenzyme activity were quantitative only with the caudate-putamen complex, midbrain, pons and medulla oblongata exhibiting relatively high content of the three isoenzymes and the cerebral cortex and olfactory bulb possessing weak isoenzyme activities. Intermediate levels of isoenzyme activities were observed in the cerebellum and hippocampus. In all areas examined, the relative percentage values for each isoenzyme remained constant. AChE isoenzymes from the forebrain, brain stem and cerebellum of 15- and 30-day-old rats appeared to have identical patterns. In brain stem, no quantitative differences could be detected in the isoenzyme activities between 15 and 30 days of age. At both ages, the isoenzymes of male and female rats did not show any qualitative differences. The single cholinesterase (EC 3.1.1.8) isoenzyme which could be identified in brain stem supernatants of 30-day-old rats was weakly reactive and appeared to have the same relative mobility as the major acetylcholinesterase zone, zone 3. Acetylcholinesterase isoenzymes failed to demonstrate any differential response toward varying concentrations of inhibitors and to changes in pH. While there were basic similarities in the acetylcholinesterase and cholinesterase isoenzyme patterns of brain, serum, liver, skeletal muscle and intestine, brain alone exhibited a marked preponderance of the acetylcholinesterase isoenzyme zone 3.     

Isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids. cDNA cloning, expression, and comparison of active sites.

Horse liver alcohol dehydrogenase occurs as isoenzymes: E is active on ethanol but not steroids; S is active on ethanol and steroids. The cDNAs for these isoenzymes were cloned; both were 1.8-kilobase long and contained complete coding sequences. Both enzymes were expressed in Escherichia coli, and the purified proteins had properties similar to those of the natural enzymes. The amino acid sequence deduced from the open reading frame of the E-type cDNA agreed with the amino acid sequence of the E isoenzyme determined by protein sequencing and x-ray crystallography. When compared with the E-type cDNA, the coding region of the S-type cDNA contains 24 substitutions and 3 deletions, giving rise to an amino acid sequence for the S. isoenzyme that differs from that of the E isoenzyme at 10 positions: nine conservative substitutions and one deletion, of Asp-115. These changes can be accommodated in the three-dimensional structure of the E isoenzyme, and models of the E and S isoenzymes complexed with a 3 beta-hydroxy-5 beta-steroid were built. The modeling shows that Leu-116 apparently sterically hinders binding of steroids in the E isoenzyme, and deletion in the S isoenzyme of Asp-115 moves Leu-116 and relieves the hindrance. The human gamma and rat liver enzymes are also active on steroids, but they have a different constellation of amino acid residues in the substrate pocket. Thus, there are multiple bases for the activity on steroids.     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Isolation and characteristics of lipoxygenase isoenzyme from pea seeds]

The individual isoenzyme lipoxygenase-2, a constituent of the heterogeneous lipoxygenase system (EC 1.13.11.12) which catalyzes coupled oxidation of beta-carotene in the presence of linoleic acid, was isolated from pea seeds and its properties were characterized. The isoenzyme has been proved to be homogeneous; some of its kinetic properties, the amino acid composition and the subunit structure have been investigated.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Structure of the O-acetylserine sulfhydrylase isoenzyme CysM from Escherichia coli

The enzyme O-acetylserine sulfhydrylase participates in the biosynthesis of l-cysteine in bacteria and plants. The structure of isoenzyme B (CysM) from Escherichia coli was established in a hexagonal crystal form at 2.7 A resolution (wild-type) and in a merohedrally twinned tetragonal crystal form at 2.1 A resolution (surface mutant). Structural superpositions revealed the variations with respect to isoenzyme A (CysK) and explained the different substrate specificities. A geometric model of the reaction catalyzed by CysM is proposed. Both isoenzymes are used for the production of l-amino acid derivatives as building blocks for the synthesis of peptides and peptidomimetic drugs. Since the structure of CysM revealed a remarkable main chain variation at the active center, it constitutes a further starting point for engineering mutants with novel substrate specificities.