Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Stål

The major insecticide resistance mechanism in the brown planthopper Nilaparvata lugens involves overproduction of esterases. Esterases purified from a resistant strain appeared as a ladder of bands on isoelectric focussing (IEF) gels from pI 4.7 to 5.0. Two-dimensional electrophoresis showed that isozymes ranged in size from 66 to 68 kDa with those of lower pI being apparently smaller. All isozymes detected by two-dimensional electrophoresis were glycosylated. N-glycosidase A reduced the number of isozymes on IEF to two, with increased pI and an increased molecular weight of 69 kDa. No O-linked glycans were detected. Deglycosylation had no effect on esterase activity, hence glycosylation is not involved in active site conformation. As N-glycosidase F completely deglycosylated the esterases, none of the glycans has an alpha1,3-bound core fucose. Reactivity with the lectins GNA, MAA and DSA, combined with differential cleavage of N-linked glycans with endoglycosidases F1 and F2, indicated that terminally linked mannose is present in high mannose and/or hybrid type glycans and that terminally linked sialic acid and galactose-beta(1-4)-N-acetylglucosamine are present in biantennary complexes. Neuraminidase treatment had the same effect on pI of isozymes as complete deglycosylation. Therefore, the majority of the heterogeneity of elevated esterases on IEF is due to differential attachment of sialic acid to glycans of the two proteins.     

Two distinct 4-hydroxynonenal metabolizing glutathione S-transferase isozymes are differentially expressed in human tissues

The two previously reported human glutathione S-transferase isozymes, hGST5.8 and hGSTA4-4, have been suggested to be similar because of their comparable activities toward 4-hydroxynonenal-GSH conjugation. Here, we demonstrate that hGST5.8 and hGSTA4-4 are distinct. Antibodies raised against hGSTA4-4 did not recognize hGST5.8, and antibodies raised against mouse GSTA4-4 that cross-react with hGST5.8 did not recognize hGSTA4-4. The pI value of hGSTA4-4 was found to be 8.4, as opposed to the pI value of 5.8 for hGST5.8. The two isozymes are differentially expressed in human tissues and there are significant differences in their kinetic properties. While both isozymes showed a strong expression in liver and testis, hGSTA4-4 was not detected in brain where hGST5.8 was present. In the pancreas, a strong expression of hGST5.8 was observed while hGSTA4-4 was barely detectable in this tissue.     

Aldehyde dehydrogenase of the Mongolian gerbil, Meriones unguiculatus.

The aldehyde dehydrogenase (Aldehyde:NAD(P) oxidoreductase E.C. 1.2.1.3. and 1.2.1.5) phenotype in several tissues of the Mongolian gerbil, Meriones unguiculatus, has been established. The tissue distribution of gerbil aldehyde dehydrogenase is similar to that of the rat, with liver possessing the majority of the aldehyde dehydrognease activity. Male kidney and testis possess significantly more activity than female kidney and ovary. The substrate and co-enzyme specificity of gerbil liver aldehyde dehydrogenase is also similar to that of rat and mouse liver. Gel isoelectric focusing resolves one major gerbil liver aldehyde dehydrogenase isozyme at pI 5.3. Mouse liver is resolved into two major isozymes at pIs 5.3 and 5.6 and rat liver aldehyde dehydrogenase into one major isozyme at pI 5.4. Gerbil liver aldehyde dehydrogenase is functional over a broad pH range with an optima at pH 9.0. Rat and mouse liver aldehyde dehydrogenase possess sharp pH optima at pH 8.5.     

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.     

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.     

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.     

Multiple molecular forms of the gibberellin-induced alpha-amylase from the aleurone layers of barley seeds

A class of plant growth regulators, gibberellins, induce the synthesis of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley (Hordeum vulgare L. var. Himalaya) seeds. The purified alpha-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These alpha-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (pI) of approximately 5.8 (the high pI group). The other group's pI values are around 4.5 (the low pI group). On some gels a small amount of protein focuses between the high and low pI isozymes. These proteins comigrate with the low pI isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high pI group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low pI group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A)+ RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of alpha-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone alpha-amylase isozymes are encoded by at least two sets of structural genes.     

Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes

Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Changes in glycolytic enzyme levels and isozyme expression in human lymphocytes during blast transformation.

The levels of each of the glycolytic enzymes were observed to exhibit a parallel increase of 200 to 300% when human lymphocytes were stimulated to undergo blast transformation. A series of electrofocusing and electrophoretic studies was utilized to assess the isozyme distribution of the glycolytic enzymes during blastogenesis. Hexokinase (pI = 7.40), glucosephosphate isomerase (pI = 9.35), and enolase (pI = 8.30) existed as single electrophoretic components and were unchanged during blast transformation. Phosphoglycerate mutase was observed to exist as two isozymes (pI = 5.80 and 6.63), which were also unchanged by blastogenesis. Aldolase, which was present as two electrophoretic forms in lymphocytes (pI = 9.25 and 8.75), exhibited a shift in the relative content of each. In addition to the lactate dehydrogenase isozymes at pI 9.50 and 7.60 found in lymphocytes, lymphoblasts contained isozymes with pI values of 7.30, 7.05, and 5.85. Although glyceraldehyde 3-phosphate dehydrogenase was present as a single electrophoretic form (pI ? 8.0) in both lymphocytes and lymphoblasts, the association of the enzyme with actin produced electrophoretic artifacts with lower pI values. Phosphoglycerate kinase, which appeared as a single form in lymphocytes (pI = 9.00), was present as two isozymes (9.00 and 8.74) in lymphoblasts. Similarly, pyruvate kinase (pI = 8.73 and 8.50 in lymphocytes) exhibited additional isozymes (pyruvate kinase, pI = 7.60 and 5.85, and triosephosphate isomerase, pI = 5.20) as a result of cell transformation.     

Purification and biochemical properties of genetically defined malate dehydrogenase in maize

Malate dehydrogenase of maize exists in multiple molecular forms (isozymes). In strain W64A, two soluble forms (s-MDH), five mitochondrial forms (m-MDH), and two glyoxysomal forms (g-MDH) were found in etiolated seedlings. The s-MDHs and m-MDHs were prepared in highly purified form. Using these purified isozymes, experiments with reducing agents (100 mm mercaptoethanol), low pH (2.0), and high salt cocn (7.5 m guanidine-HCl), along with genetic data, have eliminated the possibility of conformational alterations as an explanation for MDH multiplicity in maize; the MDH isozymes are genetically determined. Biochemical properties for each of the seven MDH isozymes were examined. Molecular weight, pI, pH optimum, thermolability, and K_m for oxaloacetate, malate, NAD, and NADH at different pH values were determined for each isozyme. Different kinetics of substrate inhibition (oxaloacetate) and coenzyme inhibition (NAD) were observed for the different isozymes. Effects of NAD analogs, chelating agents, reducing agents, metal ions, and TCA cycle acids on the enzymatic activity of these isozymes were tested. Based on the physical and kinetic properties observed, the maize malate dehydrogenase isozymes can be classified into four groups: s-MDH¹; s-MDH²; the two most anodal m-MDHs; and the three most cathodal m-MDHs. Since strain W64A is highly inbred, our data along with our previous and simultaneous genetic analysis suggest that multiple genes are involved in the expression of maize malate dehydrogenase isozymes.     

Toad carbonic anhydrase: purification of the enzyme from erythrocytes of Bufo marinus and comparison with the enzyme activity in the urinary bladder.

1. Two forms of carbonic anhydrase, having isoelectric points of 6.1 and 5.8, were purified from erythrocytes of the toad, Bufo marinus, and the presence of a third form, pI = 5.4, was demonstrated. 2. Each of the two purified isozymes catalyzed the hydration of CO2 and the hydrolysis of nitrophenyl acetate esters at rates characteristic of Type C (or high-activity) forms of carbonic anhydrase. 3. Both forms of the erythrocyte enzyme have similar molecular weights (approx 29,000), amino acid composition, sensitivity to acetazolamide, and kinetic properties. 4. The epithelium of the toad's urinary bladder also was found to contain significant amounts of carbonic anhydrase, which appears by isoelectric focusing to be indistinguishable from the enzyme isolated from the erythrocyte.     

pI(Cln) and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.

To elucidate the function of pI(Cln), its localization in subcellular organellae was investigated. A specific polyclonal anti-pI(Cln) antibody detected the soluble 38-kDa pI(Cln) exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pI(Cln)-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pI(Cln) antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pI(Cln) was present in association with actin of a constant length: the molecular ratio of pI(Cln) to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pI(Cln). These results suggest the possibility that skeletal muscle pI(Cln) controls the length of cytosolic F-actin.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

核盘菌可溶性蛋白质及同工酶电泳研究

为了研究核盘菌的分类和生理分化问题,对供试的28个核盘菌株进行了可溶性蛋白质,芳香基酯酶和酸性磷酸酯酶同工酶电泳谱带以及紫外光吸收峰图形的试验研究,其结果,可分为五种类型。其中属于核盘菌属有四个种,即核盘菌Sclerotinia sclerotiorum(Lib.)de Bary(=Whetzelinia sclerotiorum(Lib.)Korf & Dumont),三叶草核盘菌S.trifoliorum Erikss.,细辛核盘菌(S.asari Wu et C.R.Wang和人参菌核病菌Sclerotinia sp。人参菌核病的另一分离菌和油菜菌核病的一个分离菌其谱带与灰葡萄孢(Botrytis cinerea)谱带相似,应归属于与葡萄孢属(Botrytis)相应的核盘菌。在核盘菌S.sclerotiorum种群中,南方菌系与北方菌系的可溶性蛋白质稍有差异,芳香基酯酶和酸性磷酸酯酶活性强度也稍有不同,可能存在生理分化现象。     

Characterization of the isozymes of glucuronoxylan xylanohydrolase in the presence of a native cell wall substrate

K. HAYASHI, M. INOUHE, C. AOYAGI AND D.J. NEVINS. 1996. A simple but accurate method for measuring glucuronoxylan xylanohydrolase activity was developed using coleoptile cell wall particles prepared from maize ( Zea mays L.). Two isozymes of glucuronoxylan xylanohydrolases designated as GX1 and GX2 (EC 3.2.1.136) were purified from a commercially available amylase preparation to electrophoretic homogeneity by three cation exchange chromatography steps. Upon characterization no significant differences between the two enzymes were detected: the molecular mass measured by MALDITOF mass spectrometry was 44 360 100 for GX1 and 44 370 50 for GX2 suggesting no difference in the total number of amino acid residues. Furthermore the N -terminal amino acid sequence for each of the isozymes was identical through the 37th amino acid residue. The values of pI were determined to be 9.0 for GX1 and 9.1 for GX2. The sensitivity to temperature, pH and to ionic strength was similar for both isozymes as were kinetic parameters including K _m and V _maxm No differences could be detected in substrate specificity.     

Distribution and classification of acylphosphatase isozymes

Distributions of acylphosphatase isozymes among organs of several animal species were investigated. Organ extracts of pig and chicken were treated with isozyme-specific antibodies, subjected to electrophoresis on a polyacrylamide gel, then the gel was stained for acylphosphatase activity. Both animals showed three activity bands; one band was named common type isozyme because of its wide distribution in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte, and the other two bands were named muscle type isozymes because of their localization in skeletal muscle. This classification was supported by selective and quantitative reactions of the isozymes to the isozyme-specific antibodies. Because the two bands of the muscle type have the same amino acid sequence and differ only in modifications on an -SH group, it is suggested that pig and chicken have only the two major types of acylphosphatase. This conclusion was supported by similar experiments on dog, human, rabbit, and pigeon.