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PubMed2003

Structural study of the C2 domains of the classical PKC isoenzymes using infrared spectroscopy and two-dimensional infrared correlation spectroscopy.

The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.

Amino Acid Sequence↗
PubMed1998

Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

Animals↗
PubMed1997

Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymes in lung, colon and liver carcinomas.

We have compared the levels of phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activities and the distribution of their isoenzymes in normal colon, liver and lung tissues, and in colon, liver and lung adenocarcinoma, lung squamous cell carcinoma and lung carcinoid. All tumours presented higher phosphoglycerate mutase and enolase activities and lower 2,3-bisphosphoglycerate phosphatase activity than the normal tissues. No changes were observed in the phosphoglycerate mutase isoenzyme patterns analysed by cellulose acetate electrophoresis. All specimens contained mainly type BB isoenzyme, traces of type MB isoenzyme and no type MM isoenzyme. However, the tumours had decreased levels of 2,3-bisphosphoglycerate mutase and 2,3-bisphosphoglycerate mutase-phosphoglycerate mutase hybrid enzyme. Determined by agarose gel electrophoresis, alpha alpha-enolase was the isoenzyme predominant in normal lung, colon and liver tissue, although alpha gamma- and gamma gamma-enolase were also present in all tissues. In colon, liver and non-endocrine lung tumours, the proportions of alpha gamma- and gamma gamma-enolase decreased. In contrast, in carcinoid tumours of the lung, the proportions of these isoenzymes increased.

Adenocarcinoma↗
PubMed1972

Purification and comparative properties of isoenzymes of nicotinamide-adenine dinucleotide phosphate-isocitrate dehydrogenase from rat heart and liver.

1. Rat liver and heart major isoenzymes of NADP-isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg(2+) and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30 degrees C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu(2+). 5. The Michaelis constants with 3-acetylpyridine-adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.

Animals↗
PubMed1973

Hydrolysis of GM1-ganglioside by human liver beta-galactosidase isoenzymes.

1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.

Ceramides↗
PubMed1976

Adenosine 3':5'-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.

Animals↗
PubMed1978

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.

Adenosine Monophosphate↗
PubMed1988

Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.

Amino Acid Sequence↗
PubMed1998

Glutathione S-transferase of goat lens: evidence for expression of only class mu isoenzymes.

PURPOSE: It has been suggested that glutathione S-transferases (GSTs) of the mammalian ocular lens detoxify various toxicants and cataractogens; therefore, it provides protection against cataractogenesis. In this study the GST isoenzymes of goat lens have been purified and characterized. METHODS: The goat lens homogenate was subjected to affinity chromatography over GSH-linked epoxy activated Sepharose 6B. The isoenzymes of the GST were separated by chromatofocusing on a Mono-P column using FPLC. RESULTS: Two GST isoenzymes, GST 7.2 and GST 6.6, were purified to apparent homogeneity from goat lens. GST 7.2 appeared to be a heterodimer of Mr. 26,500 and Mr. 25,000 subunits, the GST 6.6 showed a homodimeric subunit structure of Mr. 25,000. Western blotting and the N-terminal region amino acid sequence analysis indicated that both isoenzymes of goat lens belong to the mu-class GST. CONCLUSION: Although they share varying degrees of structural correlation, the two isoenzymes of goat lens seem to be the products of two distinct genes. The isoenzyme expression pattern of GST in goat lens is similar to bovine lens, which also contains two isoenzymes belonging only to GST mu.

Animals↗
PubMed2006

Increased serum lactate dehydrogenase isoenzymes in Ph-negative chronic myeloproliferative diseases: a metabolic adaptation?

We evaluated the significance of lactate dehydrogenase (LDH) isoenzymes in chronic myeloproliferative disorders (CMDs) by studying LDH isoenzymes in the serum of patients with secondary polycythemia (SP), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) in different disease status. LDH activity and isoenzymes were evaluated retrospectively in serum samples from four groups of patients and compared with a control group. LDH activity and isoenzyme distributions of patients with SP and PV did not reveal significant variations with respect to controls. In the ET and IMF group LDH isoenzyme revealed significant variations: IMF showed significant increase of LDH2 and significant reduction of LDH5 isoenzyme, whereas ET showed significant decrease in LDH1 and increase of LDH3. These data suggest that LDH isoenzyme patterns may be a useful marker of CMDs, but this enzymatic pattern could be expression of a metabolic adaptation.

Adaptation, Physiological↗
PubMed2003

Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

Arachis↗
PubMed1988

Expression of glutathione S-transferase isoenzymes in human small cell lung cancer cell lines.

A glutathione S-transferase (GST) isoenzyme having common antigenicity to rat placental form (GST-P) and human placental form (GST-pi) has recently been suggested may be a marker of carcinogenesis. In the present study we have investigated the expression of this isoenzyme in three small cell lung cancer cell lines in order to determine whether or not this isoenzyme can be used as a general marker of carcinogenesis. GST activity towards 1-chloro-2,4-dinitrobenzene in two of the cell lines (NES and NOC-361) was moderately higher than that in normal human lung, but this activity was markedly suppressed in one of the cell lines (NCI-H69). Quantitation of the GST isoenzymes in the tumors grown in nude mice by injecting these cell lines also revealed a moderate increase of GST-pi-type isoenzyme in NES and NOC-361 and its suppression in NCI-H69. Immunocytochemical localization studies with these tumors using antibodies raised against GST-pi also indicated a drastic decrease of GST-pi-type isoenzyme in NCI-H69 and this finding was confirmed by Western blot studies. These results suggest that GST-pi, or the isoenzyme(s) having similar immunological nature to GST-pi, cannot be used as the general markers of neoplastic states.

Animals↗
PubMed1978

Multiple effects of glucocorticoids and hyperosmolality on isoenzyme expression in cultured KB cells.

The KB cell line, though indicted as a HeLa-contamined line, is a useful in vitro model for the study of the regulation of isoenzyme expression. KB cells produced three isoenzymic forms of alkaline phosphatase. When KB cells were grown in the presence of prednisolone and/or in hyperosmolar medium, the total enzyme activity was reduced. This change in activity was coupled with specific alterations in the proportion of each isoenzyme. The slow-moving form (identified biochemically and immunologically as the heat-stable, placental, Regan isoenzyme) was substantially increased by the steroid hormone and/or hyperosmolality. The fast-moving form [identified as the intestine-like, amnion (FL) isoenzyme] was strikingly diminished by either treatment. The intermediate form (tentatively referred to as "hybrid," inasmuch as it shared properties of the other two isoenzymes) was decreased only when KB cells were grown in hyperosmolar medium containing prednisolone. These results corroborated the notion that these stimuli cause the induction of increased levels of the heat-stable Regan alkaline phosphatase only. They also point out the necessity of performing isoenzyme analysis when one is investigating the regulation of this enzyme.

Alkaline Phosphatase↗
PubMed1976

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus.

Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.

Animals↗
PubMed1994

cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.

Alternative Splicing↗
PubMed1975

Isoenzymes of p-coumarate: CoA ligase from cell suspension cultures of Glycine max.

Two isoenzymes of p-coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4, and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite. The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc-gel electrophoresis. Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5-hydroxyferulic, p-coumaric, and caffeic acids were the substrates with the lowest apparent Km values (on all the order of 1 to 4 x 10(-5) M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 x 10(-5) M) for isoenzyme 2 were obtained for caffeic, p-coumaric, m-coumaric, and o-coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p-coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.

Cells, Cultured↗
PubMed1977

Comparative study of the effect of 5' AMP and its analogs on rabbit glycogen phosphorylase b isoenzymes.

A comparative study of the effect of AMP and other nucleotides on the different rabbit glycogen phosphorylase b isoenzymes has been made: muscle, brain, liver isoenzymes, and the hybrid species between muscle and brain isoenzyme. We have studied either the direct kinetic effect of the different nucleotides or the action of the nucleotides on the b to a conversion rate. Muscle, brain, and muscle--brain isoenzymes are very strongly and very specifically activated by AMP and its analogs, whereas liver isoenzyme is not very sensitive to the nucleotides. However, muscle, brain and muscle--brain isoenzymes show slight but very interesting differences in their kinetic properties. These differences allow some speculations about the conformational state of these different isoenzymes.

Adenosine Monophosphate↗