Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Purification and Properties of an Esterase from the Yeast Saccharomyces cerevisiae and Identification of the Encoding Gene

We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae.     

Isoelectric focusing studies of human red cell esterase D: Evidence for polymorphic occurrence of a new allele EsD 7 in Japanese

Summary The isoelectric focusing study of esterase D in Japanese revealed evidence of a new polymorphic allele (EsD ⁷) which is difficult to find by conventional starch gel electrophoresis only. A comparison with the occurrence of a subdivision of EsD ² in Caucasians (EsD ⁵) suggests a remarkable difference in allele distribution of esterase D among races. Quantitative analysis showed a relatively low value of enzyme activity for this new allele. It is therefore emphasized that in addition to conventional electrophoresis, enzyme assay and further detection by isoelectric focusing are essential in analyzing the esterase D system.     

Isoenzymes of naphthyl acetate esterase in senescing leaves of Festuca pratensis

Naphthyl acetate esterase (NAE) of leaves of Festuca pratensis had an apparent MW of 55000. Five major NAE isoenzymes were resolved by gel electrophoresis. During leaf senescence the proportions of these isoenzymes altered and two novel isoenzymes became active. Cycloheximide applied to leaves delayed and diminished the responses of NAE isoenzymes during senescence. The two novel NAEs were similar in MW and substrate affinity to pre-existing NAEs. Partially-purified NAE had no cholinesterase, carboxypeptidase, ethyl acetate esterase or ethyl butyrate esterase activity. Lack of inhibition by eserine, PCMB and organophosphorus insecticide classified these enzymes as acetylesterases.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.     

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).     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Variation and distribution of esterase isozymes inHeteranthelium andTaeniatherum of the tribe triticeae,gramineae

Esterase isozyme variations ofHeteranthelium piliferum, Taeniatherum asperum andT. crinitum collected in Iraq, Turkey and Iran were analysed by gel isoelectric focusing. InH. piliferum two types of esterase zymogram, H1 and H2, were found. It was demonstrated that a pair of allelic genes, which is shown in the heterozygotes, controls the difference between type H1 and H2. Two types of esterase zymogram, T1 and T2, were observed in two species ofTaeniatherum. The majority of strains having type H1 ofH. piliferum and type T1 ofT. crinitum was distributed in the highly elevated plateau of the Anatolian and Iranian highlands, while strains with H2 and T2 of these two species were found in the western foot-hills of the Zagros Mountains, the Tigris basin of Mesopotamia and the central Anatolian plateau.     

Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study

We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

High-Resolution Polyacrylamide Gradient Gel Electrophoresis (PGGE) of Isoenzymes from Five Naegleria Species

ABSTRACT. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis , two N. lovaniensis , one N. jadini , two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

Hormonal regulation and distribution of peroxidase isoenzymes in the Cucurbitaceae

Ethylene enhanced the levels of peroxidases in the roots, stems, leaves, and cotyledons of 2-week-old cucumber Cucumis sativus cv Poinsett 76 seedlings. Antibodies to the isoelectric point (pl) 9 and pl 4 isoenzymes were used in a radial immuno-diffusion assay to demonstrate that ethylene induced similar peroxidases in other cultivars of C. sativus, other species of Cucumis and other genera of Cucurbitaceae. Examination of ethylene-induced peroxidases, using isoelectric focusing gels, demonstrated the presence of a series of other peroxidases, mostly slightly acidic, whose isoelectric focusing pH was approximately 6. These pl 6 peroxidases were partially purified on a cation exchange column. Ouchterlony double diffusion gels indicated that these proteins cross-reacted with antibodies to both the pl 9 and pl 4 peroxidase. The data presented here suggest that the induction of peroxidase isoenzymes during ethylene-induced senescence is a common response in this family of plants. In addition, antibody and isoelectric focusing studies indicate that both acidic and basic peroxidase are highly conserved in members of this family.     

Genetic characterization of esterase 28 (ES-28) of the house mouse

The genetics of esterase-28, the major esterase of cauda epididymidis of the house mouse, has been studied after separation by polyacrylamide gel electrophoresis and isoelectric focusing. Four phenotypes are distinguished. Segregation ofEs-28 in two backcross series indicated linkage to Es-1, Es-9, and Es-22. The Es-28 locus was placed into esterase cluster 1 on chromosome 8.This work was supported by the Deutsche Forschungsgemeinschaft.This is communication No. 69 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Neurotoxic esterase: gel filtration and isoelectric focusing of carboxylesterases solubilized from hen brain

Carboxylesterase activity (EC 3.1.1.1) of hen brain including neurotoxic esterases NTEA and NTEB is solubilized from lyophilized lipid-extracted brain material by the use of n-octylglucoside. The solubilized enzymes are subjected to free isoelectric focusing, six carboxyl - esterase activity peaks are obtained. By gel filtration on Sephacryl S-300 neurotoxic esterases are separated from carboxylesterase isoenzymes V and X. The molecular weight of the neurotoxic esterases is estimated to be 1.8 X 10(6).     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

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.     

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as though they were ~ M_r 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 m guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.