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PubMed1985

Electrophoretic studies on enzymes of diploid and triploid Paragonimus westermani.

An enzyme analysis of diploid and triploid Paragonimus westermani was conducted using starch gel electrophoresis. In total, 16 enzymes, probably encoded by 18 loci, were studied for 3 populations of the diploid form sampled from 2 localities, and 4 populations of the triploid form from 4 localities. Comparison of the enzymes of the triploid and the diploid digeneans showed 5 different patterns: diaphorase (EC 1.6.2.2), glutamic-oxaloacetic transaminase (EC 2.6.1.1), hexokinase (EC 2.7.1.1), leucylglycylglycine aminopeptidase (EC 3.4.1.3), and phosphoglucomutase (EC 2.7.5.1). On the basis of the numbers of bands and their patterns, all individuals of the triploid are probably heterozygous at each of these 5 loci and homozygous at the remaining 13 loci. The occurrence of fixed heterozygotes found in triploid populations cannot be easily explained by only a single mutation. It is suggested that the variability may have been introduced by hybridization with a different sub-species or a closely related species and may, thus, have been maintained since the time of the origin of triploids.

Aminopeptidases↗
PubMed1995

Differentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing.

Isoenzyme typing was used to study a number of oocyst isolates of Cryptosporidium parvum from different geographical locations and of human or animal origin. All isolates showed identical enzyme motility when glucose phosphate isomerase (GPI; 23 isolates tested) or lactate dehydrogenases (LDH; 20 isolates tested) was assayed. However, two isoenzyme forms were observed with phosphoglucomutase (PGM; 9 animal isolates showed one form, while 8/9 human isolates showed a second form) and hexokinase (HK; 4 human isolates showed one form and 6 animal isolates showed a second form). Thus, PGM and HK each exhibit 2 isoenzymes corresponding to 2 parasite populations associated with separate hosts. The data from this study, plus supportive evidence obtained by different methods and by independent researchers, lend support to the hypothesis that separate cycles of transmission of C. parvum may exist within human and animal hosts.

Animals↗
PubMed1993

Characterization of Megatrypanum trypanosomes from European Cervidae.

Megatrypanum trypanosomes have been isolated from a number of different European Cervidae, but on the basis of morphology it has not been possible to define the species to which these isolates belong. We isolated Trypanosoma (Megatrypanum) theileri from 10 cattle, and Megatrypanum trypanosomes from 11 fallow deer (Cervus dama), 9 red deer (Cervus elaphus), and 4 roe deer (Capreolus capreolus) by blood culture on a biphasic medium (NNN agar slopes). Trypanosomes were propagated in Schneider's Drosophila medium and characterized by isoenzyme analysis and molecular karyotyping. Isocitrate dehydrogenase and phosphoglucomutase were visualized after starch gel electrophoresis of trypanosome lysates. By cluster analysis of this data all isolates from deer were clearly separated from the T.(M.) theileri isolates from cattle. Isolates from roe deer were different not only from T. (M.) theileri but also from the other deer isolates. Isolates from fallow deer and red deer were grouped together. Thus, there are probably at least two different species of Megatrypanum trypanosomes in the three Cervidae. One parasitizing roe deer, the other, apparently less host specific species, infecting red deer and fallow deer. Separation of the chromosomes of Megatrypanum trypanosomes by pulsed-field gradient gel electrophoresis (PFGE) showed that each isolate contained a large number (> 18) of chromosomes ranging in size from 300 to > 2200 kb. The molecular karyotypes were similar for all isolates, although no isolate was identical to another.

Animals↗
PubMed1991

Phosphorylated thiosugars: synthesis, properties, and reactivity in enzymatic reactions.

A number of phosphorylated thiosugars have been prepared and tested as substrates for metabolic reactions. 6-Thioglucose-6-P is readily synthesized by reaction of 6-tosylglucose with trisodium thiophosphate at pH 10 in aqueous solution; the product has only sulfur between carbon and phosphorus. When ethyl glycerate is tosylated and treated similarly with thiophosphate, a 5:1 mixture of 3-thioglycerate-3-P and the 2-isomer is formed. 6-Thioglucose-6-P is converted by glycolytic enzymes to triose phosphates, 3-thioglycerol-3-P and 3-thioglycerate-3-P, and is oxidized by enzymes of the hexose monophosphate shunt to 5-thioribulose-5-P, which can be converted via phosphoribulokinase and ribulose-bis-P carboxylase into 3-P-glycerate and 3-thioglycerate-3-P. For most of the non-phosphoryl-transferring enzymes there are only moderate effects on Vmax and Km. Phosphoglucoisomerase, however, is very sensitive to the sulfur for oxygen change, with Vmax decreasing 60-fold and Km increasing 15-fold. Surprisingly, phosphoribulokinase has a V/K value for 5-thioribulose-5-P that is over 3 orders of magnitude less than for ribulose-5-P. 6-Thio-glucose-6-P was found to be a substrate for several enzymes that transfer the phosphoryl group. It is as good a substrate for alkaline phosphatase as glucose-6-P, and with phosphoglucomutase it is converted to 6-thioglucose-1-P with a rate that is 11% of the rate of reaction of glucose-1-P, with a Keq value of 45.6. The free energy of hydrolysis of the phosphorylated thiol is thus -7.2 kcal/mol at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1976

The construction and testing of a simple, slow delivery-rapid quench apparatus.

A simple, inexpensive, slow delivery-rapid quench apparatus is described. The apparatus can be used to mix small volumes (about 50 mul) of equilibrium mixtures of enzyme-substrate and enzyme-product complexes with a quenching solution, ideally to inactivate the enzyme more rapidly than such complexes can be inconverted. The efficiency of the apparatus is tested by (a) injecting basic solutions of an indicator dye into acid and observing the length of the unbleached plume of dye produced at the delivery tip and (b) by forming an enzyme-substrate complex with 32P-labeled phosphoglucomutase and measuring the extent of label transfer prior to inactivation by the quenching solution. Problems that may be encountered in attempts to inactivate equilibrium mixtures of enzyme-substrate and enzyme-product complexes without producing quenching artifacts are considered.

Biochemistry↗
PubMed2004

Analysis of the substrate specificity loop of the HAD superfamily cap domain.

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.

Amino Acid Substitution↗
PubMed2004

Detailed kinetic studies of an aggregating inhibitor; inhibition of phosphomannomutase/phosphoglucomutase by disperse blue 56.

Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa. Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro. The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition. Quantitative analysis of the progress curves and preincubation experiments demonstrated that slow-binding inhibition was not occurring, however. Initial velocity kinetic studies indicated that Disperse Blue 56 was a parabolic, noncompetitve inhibitor. Progress curves for reactions in the presence of Disperse Blue 56 could be fitted very well by a model in which 2 equiv of the inhibitor bound to free enzyme or the enzyme-substrate complex. The inhibition was largely relieved by the inclusion of 0.01% Triton X-100 in the assay solutions, which has been suggested to be the hallmark for inhibition by compounds that exert their effect through aggregates [McGovern, S. L., Caselli, E., Grigorieff, N., and Shiochet, B. K. (2002) J. Med. Chem. 45, 1712-1722]. Our kinetic data appear to be consistent with either inhibition by a dimer of Disperse Blue 56 or inhibition by a Disperse Blue 56 aggregate, but the latter appears much more likely. We present a detailed analysis of the system to provide further information that may help in the recognition of inhibition through aggregation.

Anthraquinones↗
PubMed1997

Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella.

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3-10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM. Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

Acanthamoeba↗
PubMed1997

Genetic variation in some populations of the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela), in Portugal.

Genetic variation in the golden-striped salamander (Chioglossa lusitanica) was assessed in 231 individuals from four Portuguese populations by means of horizontal starch gel electrophoresis and isoelectric focusing. Three of 19 enzyme systems, representing 21 presumptive loci, were found to be polymorphic: phosphoglucomutase 1 (PGM1), peptidase B (PEPB), and peptidase D (PEPD). The observed average heterozygosity in Chioglossa lusitanica (0.027) is significantly lower than that observed for other amphibians, either urodeles or salamandrids. Differences in allele frequencies and the presence of private alleles are indicative of a high degree of population differentiation. PEPD, in particular, seems to be a diagnostic locus separating the southernmost population studied from the others.

Aminopeptidases↗
PubMed2004

Increased expression of H11 kinase stimulates glycogen synthesis in the heart.

OBJECTIVE: H11 kinase is a serine/threonine kinase preferentially expressed in the heart, which participates in cardiac cell growth and also in cytoprotection during ischemia. A cardiac-specific transgenic mouse overexpressing H11 kinase (2- to 7-fold protein increase) has been generated, and is characterized by cardiac hypertrophy with preserved function and protection against irreversible damage during ischemia/reperfusion. In this study, we tested whether H11 kinase also participates in the metabolic adaptation to cardiac hypertrophy and ischemia. METHODS AND RESULTS: A yeast two-hybrid screen using H11 kinase as a bait in a human heart library revealed a potential interaction with phosphoglucomutase (PGM), the enzyme converting glucose 6-phosphate into glucose 1-phosphate. Interaction between H11 kinase and PGM was confirmed by co-immunoprecipitation. To test the biochemical relevance of this interaction, PGM activity was measured in the heart from wild type and transgenic mice, showing a 20% increase of Vmax in the transgenic group, without change in KM. Glycogen content was increased proportionately to the expression of the transgene, reaching a 40% increase in high-expression transgenic mice (7-fold increase in H11 kinase protein) versus wild type (p < 0.01). Increased incorporation of glucose into glycogen was coupled to a 3-fold increase in the protein expression of the glucose transporter GLUT1 in plasma membrane of transgenic mice (p < 0.01). CONCLUSION: H11 kinase promotes the synthesis of glycogen, an essential fuel for the stressed heart in both conditions of overload and ischemia. Therefore, H11 kinase represents an integrative sensor in the cardiac adaptation to stress by coordinating cell growth, survival and metabolism.

Animals↗
PubMed1986

Isoenzyme studies of Giardia lamblia isolated from symptomatic cases.

Strains of Giardia lamblia were isolated from symptomatic cases of giardiasis and axenized in the laboratory. Electrophoretic mobility patterns of four enzymes, viz., EC 5.3.1.9 glucose phosphate isomerase (GPI); EC 1.1.1,4.0.L-malate; NADP+ Oxidoreductase (Oxaloacetate decarboxylating) (ME); EC 2.7.5.1 phosphoglucomutase (PGM); and EC 2.7.1.1 hexokinase (HK) of the lysates prepared from these isolates were studied using starch-gel. Based on differences in mobility patterns of PGM and HK, the four strains studied could be grouped into three different isoenzyme types (Zymodemes). ME mobility was identical in all the four strains. Some relative difference was seen in the mobility of GPI, though the pattern of mobility was similar in all the strains.

Adolescent↗
PubMed2005

Long-term stability of allozyme frequencies in a wood lemming, Myopus schisticolor, population with a biased sex ratio and density fluctuations.

Wood lemming (Myopus schisticolor) populations are characterized by female biased sex ratios and cyclic variations in population size. Both of these characteristics are assumed to reduce genetic variation and thus affect the evolutionary adaptation of the species. We addressed these questions by studying the genetic structure of a wood lemming population from eastern Finland by isozyme markers during a 21-year period, which corresponds to 40-50 generations. Contingency tests showed that genotypic proportions conformed to Hardy-Weinberg equilibrium in each of the four sampling years. Among the temporal replicates, allele frequencies differed most by 0.14 and were not significant. Genetic variation was also stable and fairly high with a mean observed heterozygosity of H = 0.057. Variability in the Heinavesi population was higher than previously reported in wood lemming. The difference was mainly caused by variation at a phosphoglucomutase locus that was monomorphic in earlier studies. Significant linkage disequilibrium was observed in three of the comparisons but the disequilibrium did not appear consistently in all years. This pattern was also evidenced by the variance components, which indicated that selection favoured for specific allele pairs only in few subsamples.

Animals↗
PubMed1967

Divergent effects of actinomycin d on cortisol and on glucose stimulation of glycogenesis in mouse liver.

1. The administration of cortisol and of other glucocorticoid steroids to starved mice produced an increase in liver glycogen content, an elevation of glycogen-synthetase activity and a predominantly particulate localization of both phosphorylase and glycogen-synthetase enzymes. 2. Three daily doses of actinomycin D caused a marked glycogen depletion, a significant decrease in glycogen-synthetase activity, the solubilization of phosphorylase and glycogen synthetase and the following effects on the activities of various other enzymes: a decrease in UDP-glucose pyrophosphorylase and phosphoglucomutase, an increase in glucose 6-phosphate dehydrogenase and no change in glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, pyruvate kinase and UDP-glucose dehydrogenase. 3. Glucose ingestion, but not cortisol administration, reversed the effects of actinomycin D on liver glycogen content and on the activities of phosphorylase and glycogen synthetase.

Animals↗
PubMed1967

Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning.

1. The enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, UDP-glucose pyrophosphorylase, phosphofructokinase, ATP-citrate lyase and acetyl-CoA carboxylase have been assayed in rat mammary glands in various stages of involution after hypophysectomy and weaning. 2. After hypophysectomy all seven enzymes decline in activity over a 12-16hr. period but the extent of the decline varies, with acetyl-CoA carboxylase becoming almost totally inactive, ATP-citrate lyase and phosphofructokinase showing a large decrease, and the remaining enzymes a less marked decline. 3. Within 24hr. of removing the litter a change in the pattern of enzyme activity is found very similar to that after hypophysectomy. 4. The significance of these results is discussed in relation to the endocrine control of mammary gland metabolism and the mechanisms of involution.

Animals↗
PubMed1969

The pentose phosphate pathway of glucose metabolism. Influence of a growth-hormone-secreting pituitary tumour on the oxidative and non-oxidative reactions of the cycle in liver.

1. Measurements were made of the activities of enzymes of the pentose phosphate cycle, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase, as well as of the related or competing enzymes glucokinase, hexokinase, phosphoglucose isomerase and phosphoglucomutase, in control rats and in rats bearing the growth-hormone- and prolactin-secreting pituitary tumour MtTW5, to study the effect of high endogenous concentrations of growth hormone on this pathway in liver. 2. There was a twofold increase in liver weight. Glucokinase activity/g. of liver decreased to half the control value in the experimental group, although on a total liver basis it remained unchanged. Hexokinase activity increased in parallel with the liver weight, so that the total activity was doubled in rats with a high endogenous concentration of growth hormone. No differences in response were found between heat-stable and heat-labile forms of hexokinase. 3. The activity/g. of liver of the two oxidative enzymes of the pathway decreased slightly in the experimental group, but this was offset by the increase in liver weight, and the resultant effect was a 50% increase in the total activity. 4. Of the non-oxidative enzymes of the cycle the most marked increase on a total liver basis was in ribose 5-phosphate isomerase activity, to 2.5 times the control value. Ribulose 5-phosphate epimerase activity showed the smallest increase. Transketolase and transaldolase activities were also increased. The latter is the rate-limiting enzyme of the non-oxidative reactions of the cycle in these animals. 5. The results are discussed in relation to the glycolytic pathway and synthesis of glycogen, and more particularly to the increased requirement for ribose 5-phosphate for RNA synthesis.

Animals↗
PubMed1970

Variations in the activity of several enzymes in the mammary glands of non-pregnant, pregnant and lactating rabbits.

1. The enzymes phosphofructokinase (EC 2.7.1.11), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) were assayed in rabbit mammary glands at various stages of the pregnancy-lactation cycle. 2. The activities of all enzymes were low during pregnancy and, with the exception of phosphofructokinase, in non-pregnant animals. Two- to ten-fold increases in enzyme activities occurred over the first 20 days of lactation. Although milk yield was considerably decreased, the enzyme activities remained elevated in late lactation (45 days after parturition). 3. These findings are discussed in relation to mammary-gland metabolism and compared with similar observations previously made on ruminants and other small mammals.

Animals↗
PubMed1971

Studies on the structure and metabolism of glycogen in some abnormal human infants.

1. The liver and muscle tissues of 14 human anencephalic babies were examined for glycogen content and structure, and for the activities of several glycogen-metabolizing enzymes. 2. In both tissues glycogen content increased with gestation age; the muscle glycogens had a slightly but significantly lower degree of branching than the corresponding liver glycogens. 3. All the expected glycogen-metabolizing enzymes were present; acid maltase activities were higher and phosphoglucomutase activities were lower than the results reported for human adult tissues. Glucose 6-phosphatase activity increased significantly with gestation age.

Anencephaly↗
PubMed1973

Metabolic adaptations during lactogenesis. Fatty acid and lactose synthesis in cow mammary tissue.

1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days post partum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities of relevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and alpha-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (alpha-lactalbumin) serving a similar function in lactose biosynthesis.

Acetates↗