Differences In Enzyme Content Research Articles

Hybridization in Sunfish Influences the Muscle Metabolic Phenotype

Hybridization has the potential to exert pleiotropic effects on metabolism. Effects on mitochondrial enzymes may arise through incompatibilities in nuclear- and mitochondrial-encoded subunits of the enzyme complexes of oxidative phosphorylation. We explored the metabolic phenotype of bluegill (Lepomis macrochirus), pumpkinseed (Lepomis gibbosus), and their unidirectional F(1) hybrids (male bluegill × female pumpkinseed). In hybrids, glycolytic enzyme activities were indistinguishable from (aldolase, pyruvate kinase) or intermediate to (lactate dehydrogenase, phosphoglucoisomerase) parentals, but complex IV activities aligned with pumpkinseed, both 30% lower than bluegill. In isolated mitochondria, the specific activities of complexes I, II, and V were indistinguishable between groups. However, both complex III and IV showed indications of depressed activities in hybrid mitochondria, though no effects on mitochondrial state 3 or state 4 respiration were apparent. The patterns in complex IV activities were due to differences in enzyme content rather than enzyme V(max); immunoblots comparing complex IV content with catalytic activity were indistinguishable between groups. The sequence differences in complex IV catalytic subunits (CO1, CO2, CO3) were minor in nature; however, the mtDNA-encoded subunit of complex III (cytochrome b) showed eight differences between bluegill and pumpkinseed, several of which could have structural consequences to the multimeric enzyme, contributing to the depressed complex III catalytic activity in hybrids.

Neuroanatomical specificity of sex differences in expression of aromatase mRNA in the quail brain

In birds and mammals, aromatase activity in the preoptic-hypothalamic region (HPOA) is usually higher in males than in females. It is, however, not known whether the enzymatic sex difference reflects the differential activation of aromatase transcription or some other control mechanism. Although sex differences in aromatase activity are clearly documented in the HPOA of Japanese quail ( Coturnix japonica), only minimal or even no differences at all were observed in the number of aromatase-immunoreactive (ARO-ir) cells in the medial preoptic nucleus (POM) and in the medial part of the bed nucleus striae terminalis (BSTM). We investigated by in situ hybridization the distribution and possible sex differences in aromatase mRNA expression in the brain of sexually active adult quail. The distribution of aromatase mRNA matched very closely the results of previous immunocytochemical studies with the densest signal being observed in the POM, BSTM and in the mediobasal hypothalamus (MBH). Additional weaker signals were detected in the rostral forebrain, arcopallium and mesencephalic regions. No sex difference in the optical density of the hybridization signal could be found in the POM and MBH but the area covered by mRNA was larger in males than in females, indicating a higher overall expression in males. In contrast, in the BSTM, similar areas were covered by the aromatase expression in both sexes but the density of the signal was higher in females than in males. The physiological control of aromatase is thus neuroanatomically specific and with regard to sex differences, these controls are at least partially different if one compares the level of transcription, translation and activity of the enzyme. These results also indirectly suggest that the sex difference in aromatase enzyme activity that is present in the quail HPOA largely results from differentiated controls of enzymatic activity rather than differences in enzyme concentration.

Variance of Microsomal Protein and Cytochrome P450 2E1 and 3A Forms in Adult Human Liver

Differences in the pharmacokinetics of xenobiotics among humans makes them differentially susceptible to risk. Differences in enzyme content can mediate pharmacokinetic differences. Microsomal protein is often isolated from liver to characterize enzyme content and activity, but no measures exist to extrapolate these data to the intact liver. Measures were developed from up to 60 samples of adult human liver to characterize the content of microsomal protein and cytochrome P450 (CYP) enzymes. Statistical evaluations are necessary to estimate values far from the mean value. Adult human liver contains 52.9 ± 1.476 mg microsomal protein per g; 2587 ± 1.84 pmoles CYP2E1 per g; and 5237 ± 2.214 pmols CYP3A per g (geometric mean ± geometric standard deviation). These values are useful for identifying and testing susceptibility as a function of enzyme content when used to extrapolate in vitro rates of chemical metabolism for input to physiologically based pharmacokinetic models which can then be exercised to quantify the effect of variance in enzyme expression on risk-relevant pharmacokinetic outcomes.

Integrated biochemical, molecular genetic, and bioacoustical analysis of mesoscale variability of the euphausiid Nematoscelis difficilis in the California Current

Integrated assessment of the euphausiid Nematoscelis difficilis (Crustacea; Euphausiacea) and the zooplankton assemblage of the California Current was designed to investigate individual, population, and community responses to mesoscale variability in biological and physical characters of the ocean. Zooplankton samples and observational data were collected along a cross-shelf transect of the California Current in association with the California Cooperative Fisheries Investigations (CalCOFI) Survey during October 1996. The transect crossed three domains defined by temperature and salinity: nearshore, mid-Current, and offshore. Individual N. difficilis differed in physiological condition along the transect, with higher size-corrected concentrations of four central metabolic enzymes (citrate synthetase, hexokinase, lactate dehydrogenase (LDH), and phosphoglucose isomerase (PGI)) for euphausiids collected in nearshore waters than in mid-Current and offshore waters. There was little variation in the DNA sequences of the genes encoding PGI and LDH (all DNA changes were either silent or heterozygous base substitutions), suggesting that differences in enzyme concentration did not result from underlying molecular genetic variation. The population genetic makeup of N. difficilis varied from sample to sample based on haplotype frequencies of mitochondrial cytochrome oxidase I (mtCOI; P=0.029). There were significant differences between pooled nearshore and offshore samples, based on allele frequencies at two sites of common substitutions in the mtCOI sequence ( P=0.020 and 0.026). Silhouette and bioacoustical backscattering measurements of the zooplankton assemblage of the top 100 m showed marked diel vertical migration of the scattering layer, of which euphausiids were a small but significant fraction. The biochemical and molecular assays are used as indices of complex physiological (i.e., growth and condition) and genetic (i.e., mortality) processes; the bioacoustical observations provide insight into the ecosystem context for the single-species measurements. All data are intended for integration into predictive models of secondary production and biomass concentration in the ocean.

Enzyme adaptation along a heterothermic tissue: the visceral retia mirabilia of the bluefin tuna.

We measured enzyme activities along a heterothermic tissue, the visceral retia mirabilia of the bluefin tuna, to test current theories of enzyme temperature adaptation. The heterothermic tissue model is ideal for the study of fundamental temperature adaptation because it eliminates confounding effects of whole animal acclimation. Enzymes were measured at six positions along the rete at four temperatures (15, 20, 25, and 30 degrees C). Five enzymes (aspartate aminotransferase, citrate synthase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, and pyruvate kinase) exhibited a significant positive compensatory effect, with activity at the cold end of the rete 1.2-3.1 times higher than at the warm end. Two enzymes (alanine aminotransferase and lactate dehydrogenase) exhibited no significant compensation. On the basis of activation energies of enzymes along the rete, differences in activity were due to differences in enzyme concentration and not isozymes or enzyme modification. Analysis of the compensatory responses of the enzymes in light of their thermal sensitivities leads us to conclude that the pentose phosphate shunt is especially enhanced at the cold end of the rete.

Interindividual variation in the enzymatic 15-keto-reduction of 13,14-dihydro-15-keto-prostaglandin E1 in human liver and in human erythrocytes.

The therapeutic response to PGE1 is highly variable, and a contribution by variable formation of its active tertiary metabolite PGE0 is in question. Hence, the objective of this study was to assess the person-to-person variation of the reduction of the inactive intermediate metabolite 15-KD PGE1 by human liver and human erythrocytes in forming the active metabolite PGE0. Source of enzyme was lysed erythrocytes from 29 donors, and a bank of 37 donor livers including specimens from 15 children. Tritium-labelled 13,14-dihydro-15-keto-prostaglandin E1 (15-KD PGE1) was used at low nanomolar concentrations and found to be converted almost exclusively to the more polar compound 13,14-dihydro-prostaglandin E1 (PGE0) by an NADPH-dependent carbonyl reductase. The identity of the product PGE0 was established by comparison of its chromatographic and mass spectral characteristics with authentic PGE0. Lysed erythrocytes had readily measurable enzymatic activity; differences between the preparations from 29 subjects were very small with only a twofold range of variation. In contrast to lysed erythrocytes, intact erythrocytes did not catalyse the reaction so that the erythrocyte activity should be medically immaterial. 15-KD PGE1 15-ketoreductase activity of liver cytosol averaged 61.1 fmol.min-1.mg-1 protein in preparations from 37 human livers. Individual activities varied over an almost tenfold range, with indications of a non-normal distribution. Kinetic studies of selected specimens showed substantially different Vmax values but indistinguishable kM values, suggesting that the individual variation in 15-KD PGE1 15-ketoreduction is the result of differences in enzyme concentration rather than of structural enzyme variations. The activity in 15 livers from children was significantly lower than in those from adults. Inhibition data suggest that both the liver and the erythrocyte enzymes belong to the class of carbonyl reductases. The variations in hepatic enzyme activity may be expected to affect the transformation of 15-KD PGE1 to the active metabolite PGE0 in vivo. The clinical significance remains to be explored.

The establishment of the hepatic architecture is a prerequisite for the development of a lobular pattern of gene expression.

We have studied the expression patterns of ammonia-metabolising enzymes and serum proteins in intrasplenically transplanted embryonic rat hepatocytes by in situ hybridisation and immunohistochemical analysis. The enzymic phenotype of individually settled hepatocytes was compared with that of hepatocytes being organised into a three-dimensional hepatic structure. Our results demonstrate that development towards the terminally differentiated state with zonal differences in enzyme content requires the incorporation of hepatocytes into lobular structures. Outside such an architectural context, phenotypic maturation becomes arrested and hepatocytes linger in the protodifferentiated state. These features identify the foetal period as a crucial time for normal liver development and show that the establishment of the terminally differentiated hepatocellular phenotype, beginning with the differentiation of hepatocytes from the embryonic foregut, is realised via a multistep process.

Enkephalin metabolism

Inhibition of enkephalin hydrolysis by catecholamines in vitro suggested that local and/or humoral factors released during exercise might facilitate opiate responses by reducing the rate of opiate peptide inactivation. Several measures of enkephalin hydrolysis were determined in blood samples obtained from subjects designated as trained (VO2max, 64.3 +/- 1.6 ml.min-1.kg-1) and un-trained (VO2max, 37.4 +/- ml.min-1.kg-1) both at rest and after maximal exercise stress tests. Enkephalin hydrolyzing activity assessed under optimal conditions was equally distributed between plasma and intact red cells; however, hydrolysis by red cells increased dramatically following osmotic release of red cell contents. There were no apparent differences in enzyme concentration or its distribution between cells and plasma when comparing trained and untrained subjects; P greater than 0.05. There was also no statistical effect of maximal exercise on these measures in either group. However, when the sequential disappearance of enkephalin added to whole blood in vitro was evaluated, blood from trained subjects degraded the enkephalin more slowly than blood from untrained subjects and had half-lives in vitro 30-50% longer both before and after the exercise test; P less than 0.05. Since enzyme concentrations between the groups were similar, the longer half-lives suggest that circulating factors were responsible for moderating the rate of enkephalin metabolism in vivo and that these factors were more concentrated in trained subjects. This would facilitate opiate responses in trained subjects and perhaps provide them with added tolerance for the effort associated with elite performance levels.

Renal oncocytoma

Using a polyclonal antibody raised against bovine heart cytochrome c oxidase, the occurrence of this mitochondrial marker enzyme has been investigated in 63 kidney tumors (ten renal oncocytomas, 43 renal cell carcinomas and ten tubulopapillary adenomas) as well as in normal renal tissue by an immunoperoxidase method (PAP-technique). The differentiation between renal oncocytomas and mitochondria-rich carcinomas represents a problem of histopathology since these tumors have a different prognosis and require different patient managements. The strong immunoreactivity in renal oncocytomas contrasted with the much weaker reactivity in renal carcinomas and adenomas. Even mitochondria-rich (granular cell type) carcinomas exhibited only moderate staining intensity. Furthermore, single strongly stained oncocytes or small complexes were sometimes detected in normal renal tissue. The demonstration of marked differences in enzyme content between renal oncocytomas and granular cell carcinomas renders this method suitable for unequivocal distinction between these renal neoplasms. The antibody proved to be a valuable marker for detecting "true" oncocytic transformation in renal tumors and was useful in defining even single oncocytes or small oncocytic lesions.

Androgen responsiveness of proteinase F in the submandibular salivary glands of inbred mice.

Using the radioimmunoassay technique, we studied androgenic regulations of proteinase F in the submandibular salivary glands (SMG) of various mouse strains (ICR, BALB/CA, C3H/HeN, C57BL/10N, and DBA/2N). The proteinase contents in BALB/CA and DBA/2N mice were almost the same between males and females. However, prominent sex differences in enzyme content were seen in all other strains; i.e., male glands of C3H/HeN and C57BL/10N contained more proteinase F than female glands did. On the contrary, the enzyme level in the ICR was exceedingly higher in females than in males. The enzyme contents in ICR SMG increased in males following castration and decreased after androgen injection in both castrated males and normal females. On the contrary, the enzyme level in C3H/HeN was decreased by castration and increased by androgen injection. Therefore, these sex differences in ICR and C3H/HeN mice depend on androgen, though the direction of the response is opposite.

A rapid enzymatic procedure for "fingerprinting" bacteria by using pattern recognition of two-dimensional fluorescence data.

We describe a rapid fluorescence technique for bacterial "fingerprinting," based on the differences in enzyme content and activity of various bacteria. Bacterial cells are incubated with a mixture of carefully chosen fluorogenic enzyme substrates to produce fluorophores with unique emission and excitation properties. The resulting two-dimensional fluorescence spectrum of the product mixture produces a pattern characteristic of the bacterium. A Fourier-transform-based pattern recognition algorithm is used for spectral matching and for differentiating visually similar spectra. This sensitive technique is potentially applicable to differentiation and identification of bacteria in clinical laboratories.

Fenofibrate and human liver

In rodents fenofibrate shares with other triglyceride-lowering agents the potential to increase the liver peroxisome population. It was therefore of interest to look for this effect in hyperlipoproteinemic patients receiving this drug. Light and electron microscopy of liver biopsies from a group of 10 patients treated with fenofibrate and from another group of 15 receiving diet only, show no morphological difference between both groups. In contrast with the rodent data the morphometric study reveals no significant changes in the number (fenofibrate group: 7.96 10(10) cm-3; group receiving diet alone: 8.41 10(10) peroxisomes/cm3 of liver tissue) or in the size (fenofibrate group: Diameter = 0.53 +/- 0.07 micrometer--group receiving diet alone: 0.50 +/- 0.06) of peroxisomes. The difference between our results and those obtained consistently in rodents may be due to the relatively low dose in man and/or a species-dependent difference in enzyme content of liver peroxisomes, itself related to an apparent difference in the way in which lipids are handled.

The Maintenance of Different Enzyme Activity Levels in Congeneric Fishes Living at Different Depths

To determine the influence of depth-related factors on enzymic activity and, hence, on metabolism, we measured enzymic activities of white skeletal muscle and brain in two teleost fishes of the genus Sebastolobus (Scorpaenidae). These species are genetically close and have similar life histories but differ in their depth distributions. White skeletal muscle of the shallow-living species, S. alascanus, contained approximately twice the activity (measured as units per g wet weight of tissue) of lactate dehydrogenase (LDH), pyruvate kinase (PK), malate dehydrogenase (MDH), creatine phosphokinase (CPK), and citrate synthase (CS) as the white muscle of the deep-living species, S. altivelis. The CS and LDH activities exhibited body-sizerelated scaling; CS activity decreased and LDH activity rose in larger-sized specimens. The interspecific activity differences may derive either from differences in substrate turnover number (e.g., LDH) or from differences in enzyme concentration (e.g., PK). Through these two activity-regulating mechanisms, constant ratios of activity of different enzymes within the muscles of each species are maintained, while approximately twofold interspecific differences are maintained. The interspecific differences in enzymic activity are attributed to the importance of depth-related environmental factors, e.g., food availability, lack of light, and a low-energy physical environment, which may permit reduced levels of metabolism at depth. Correlated with the reduced capacity for muscle metabolism in S. altivelis is a small (9%) reduction in muscle-buffering capacity. Protein and water content did not differ in the muscle tissues of the two species. Unlike muscle, in brain tissue no interspecific differences in enzymic activity (LDH, PK, and CS) were found. This suggests that the differences in enzymic activity noted for muscle tissue reflect muscle-specific changes associated with reduced metabolic and locomotory demands at depth. Comparisons of congeneric species experiencing similar thermal regimes and having similar life history and ecological characteristics, but having different depth distributions, permit sensitive analyses of depth-related biochemical adaptations.

Galactose-1-phosphate uridyltransferase of lemurs: A comparison of four species

Erythrocyte galactose-1-phosphate uridyltransferase (Gal-1-PUT) was studied in four species of lemurs. Electrophoretic phenotypes of Lemur fulvusand Lemur macacowere indistinguishable and different from the phenotypes of Lemur cattaand Lemur variegatus,which were different from each other. Enzymatic activity of hemolysates was species specific, with that of Lemur variegatusabout twice that of either Lemur fulvusor Lemur macacoand almost three times that of Lemur catta.Only minor interspecific differences were demonstrated in pH optima and km for galactose-1-phosphate; however, thermal stability varied considerably with phenotype. Antibody inhibition studies indicated that differences in enzyme activity of hemolysates from these species are probably due to differences in enzyme concentration.

The Growth Rate of Successive Leaves of Wheat Plants in Relation to Sugar and Protein Concentrations in the Extension Zone

The growing part of a wheat leaf (the extension zone) is loocated at the leaf base and following from this it was proposed that the absolute leaf extension rate (Ler) can be partitioned into two components: the length of the extension zone (Lez) and the relative extension rate of that extension zone (Rez). Rez is an appropriate measure of the efficiency of leaf growth for comparing different leaves. This model of a wheat leaf was then used to investigate whether differences in growth rates between successive leaves on wheat plants were due to differences in hexose sugar or protein concentrations within the extension zone. Measurements were done in an irrigated field crop supplied with 0, 3, 10, or 30 kg N ha−1 per week. The mean values of Ler at 15 °C increased with leaf number and with nitrogen supply as did values for Lez. In contrast Rez at 15 °C declined from 0.9 d−1 for the first leaves to 0.3 d−1 for the flag leaf. Nitrogen supply had little effect on Rez. A separate measure of the efficiency of leaf growth, the responsiveness of Ler to temperature (measured as the slope of the temperature response curve), also decreased with leaf number by the same order as Rez and was similarly unaffected by nitrogen supply. The protein concentrations in the extension zones of the first leaves were around 40 mg g−1 fr. wt. and this declined to approximately 20 mg g−1 fr. wt for leaves emerging after tillers emerged and remained low thereafter. Protein concentrations were not correlated with the external supply of nitrogen. Hexose sugar concentrations followed a reverse pattern of increasing in the later order leaves and these also increased as nitrogen supply decreased. Both Rez and the responsiveness of Ler to temperature were positively correlated with the protein concentration, the relationship in each case being described by a rectangular hyperbola equation (P < 0.01), and negatively correlated with hexose concentrations. It was concluded that internal competition between growing points for reduced nitrogen caused the observed effects. However, differences in protein concentrations may not simply reflect differences in enzyme concentrations; rather these differences may indicate changes in some other character such as cell numbers.

Functional heterogeneity among peritoneal macrophages: II. Enzyme content of macrophage subpopulations

Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.

Formula omitted] activity in normal and chronic lymphocytic leukaemic lymphocytes

N- Acetyl-β- d-glucosaminidase (EC 3.2.1.30) activity was measured fluori-metrically in: 1. (a) lymphocytes from 20 normal donors, 2. (b) enriched B and T lymphocyte populations prepared by E rosette sedi mentation from 8 normal subjects, 3. (c) lymphocytes from 15 untreated B cell chronic lymphocytic leukaemic patients. The pH profiles and optima (4.7) were similar in all preparations. Normal B lymphocytes had higher activity than normal T lymphocytes and both these preparations and normal unfractionated lymphocytes had significantly higher activity than chronic lymphocytic leukaemic lymphocytes. The apparent Michaelis constants were similar in normal unfractionated, B enriched and T enriched lymphocytes, whereas a reduced affinity for the enzyme was observed in the leukaemic lymphocytes. The difference in enzyme content between normal and chronic lymphocytic leukaemic lymphocytes cannot therefore be explained on the basis of a high B cell percentage in patients with chronic lymphocytic leukaemia.

Some properties of the ribonuclease activities from the parotid and submandibular salivary glands of the neonatal and adult rat

The accumulation of ribonuclease activity in the perinatal submandibular gland and its decrease with adulthood was not due to the presence of inhibitors or activators and thus reflects real differences in enzyme concentration. The RNase activities of parotid and submandibular glands of the 4-day old and adult rat were purified by DEAE-Sephadex and/or by affinity chromatography and their properties were compared. RNases from the different sources were identical in their behaviour on DEAE-Sephadex chromatography, and in their pH optima, acid-stability, heat-stability and lack of activity against polyadenylic acid. The 4-day submandibular RNase was identical to the adult parotid enzyme in its electrophoretic mobility on polyacrylamide gel. Measurements were made of the relative activity of the various RNases against polycytidylic acid (poly C) versus RNA; all had greater activity against poly C. In general, considerable variability in the poly C:RNA activity ratio among different preparations precludes a confident conclusion about their similarity or difference. However, the poly C:RNA ratio of the adult submandibular gland RNase was lower than that of all of the others, and this enzyme may be different from both the RNase of the neonatal submandibular gland and that of the adult parotid gland.

Neurospora crassa glutamine synthetase. Role of enzyme synthesis and degradation on the regulation of enzyme concentration during exponential growth

The specific activity of Neurospora crassa glutamine synthetase varies according to the nitrogen source in which the organism is grown. In a poor nitrogen source such as glutamate, the specific activity of the enzyme is higher than that found in good nitrogen sources such as ammonium or glutamine. These differences in specific enzyme activity correspond to differences in enzyme concentration. The relative rates of glutamine synthetase synthesis and degradation were measured in exponential cultures grown in different nitrogen sources. The differences in enzyme concentration are explained by differences in the relative rate of enzyme synthesis.

Pyridine Nucleosidase in Bull Semen I. Breed and Sire Differences in Enzyme Concentration

The concentration of pyridine nucleosidase which cleaves the nicotinamide-ribose bond in NAD and related compounds was assayed using the cyanide addition reaction in semen samples collected by artificial vagina from 246 bulls. Among the 205 sires of recognized dairy breeds the nucleosidase concentration ranged from 0 to 1470 units/ml semen. Of the dairy sires 18% produced semen containing no nucleosidase activity. Among the 41 sires of recognized beef breeds 54% had no nucleosidase and the highest concentration was 630 units/ml semen. Among 20 sires from each of which 5-17 collections were assayed, there were significant differences in average nucleosidase concentration. There was a discontinuous distribution in nucleosidase concentration among the 169 dairy sires producing semen with measurable enzyme activity. Results for 18 dairy sires each with from 4 to 16 sons showed that sires with no seminal nucleosidase produced more sons with no enzyme or lower concentrations of enzyme than sires with higher nucleosidase concentrations. It is suggested that the concentration of the nucleosidase in bull semen is simply inherited.