Microphotometric measurement of some enzymatic activities in rabbit spermatozoa during epididymal maturation

Cytochemical quantitative measurements of isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities were made on rabbit spermatozoa collected from the testis, the different epididymal sites and the ductus deferens. These measurements were made on individual spermatozoa (at least 100 spermatozoa for each site under consideration) using a Vickers M 85 scanning microdensitometer.The activity patterns of the enzymes involved in the tricarboxylic acid cycle (ICDH, MDH) and in the respiratory chain (cytochrome oxidase) both showed a progressive decrease in the intramitochondrial oxidative metabolism from the testis to the ductus deferens. This was in contrast to LDH activity which represents the anaerobic glycolysis pathway rather than the activity of intramitochondrial LDH. The G6PDH activity could be related to those membrane modifications which the male gamete undergoes during its epididymal maturation. Potential GLDH activity was relatively intense in the spermatozoa from the testis and from the initial and distal segments of the genital tract, suggesting an intramitochondrial synthesis of enzymes such as cytochrome oxidase or ATPase.The quantitative variations of the enzymatic activities occurring during the transit of spermatozoa along the male genital tract suggested the existence of different specific interactions between the spermatozoon and the epididymal microenvironment.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Sodium dodecyl sulfate, concurrent inhibitor of several dehydrogenases

The effect of sodium dodecyl sulfate on the activity of highly purified or crystalline enzymes has been studied. The enzymes were: lactate dehydrogenase (LDH), malate dehydrogenase (MDH). isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6P-DH), lipase, alkaline phosphatase. Sodium dodecyl sulfate, always under the critical micellar concentration, shows a selective inhibitory effect. A kinetic analysis of the inhibitory action on LDH, MDH, ICDH and G6P-DH was also carried out.     

Glucose-6-phosphate dehydrogenase and malate dehydrogenase enzyme electrophoretic patterns amongst strains of Bacteroides fragilis

Fifty-two strains of Bacteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

The palmitoleate: a natural selective denaturant of enzymes

A study has been carried out in order to explain the enzyme-palmitoleate interaction. The highly purified and crystalline enzymes representative of fundamental metabolic pathways were: alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6P-DH), alkaline phosphatase. The enzyme-palmitoleate interaction was studied as a phenomenon time-independent (inhibition) and time-dependent (inactivation). Palmitoleate inhibited remarkably LDH, MDH, ICDH and G6P-DH. A kinetic analysis of the inhibitory action of palmitoleate on LDH and MDH was also carried out. Inactivation studies have shown that ADH and alkaline phosphatase are not sensitive to palmitoleate action, unlike the other enzymes. A comparison was made between the action of palmitoleate and that of a synthetic anionic detergent, sodium dodecyl sulfate (SDS).     

Enzymatic activity in crude oil contaminated rats

The activity of enzymes found in the plasma, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), and enzymes from erythrocytes, glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase, was studied in rats contaminated by crude oil. Crude oil (tube fed) contamination caused a significant increase in MDH and LDH activity 96 hr after contamination while a decrease in activity was noted in 6-6-PDH and catalase. An additional contamination (1 week after the first contamination), measured 96 hr after contamination, caused a relative decrease in MDH and LDH activity while there was a contrasting relative increase in G-6-PDH and catalase activity. After a recovery period of 3 weeks the only significant change was an increase in catalase activity.     

OESTROGEN EFFECTS ON BRAIN AND PITUITARY ENZYME ACTIVITIES

Abstract— Ovariectomized female rats were treated daily with oestradiol-17β benzoate for intervals up to one week and enzyme activities were measured in the pituitary and various brain regions. Brain regions were selected for study on the basis of their previously demonstrated content of putative oestradiol receptor sites. (1) Pituitary showed oestrogen-dependent increases in glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and lactic dehydrogenase (LDH), and no change in NADP⁺-dependent isocitric dehydrogenase (ICDH), NADP⁺-dependent malic dehydrogenase (MDH) or hexokinase (HK). MDH and ICDH were elevated in whole hypothalamus. Enzyme activities did not change significantly in whole amygdala, cerebral cortex, or hippocampus. (2) Sub-regions of the preoptic area, hypothalamus and amygdala were dissected to obtain more highly concentrated populations of cells containing putative oestrogen receptor sites. In the basomedial sub-region of hypothalamus, activities of MDH, ICDH and G6PDH were elevated by oestrogen treatment. In the corticomedial sub-region of amygdala, MDH and ICDH were elevated by oestrogen treatment. No change was observed in any of the six enzymes in medial preoptic area. (3) Increases in enzyme activities were related to the total in vivo dose of oestradiol benzoate given. (4) Hypophysectomy or adrenalectomy did not prevent the enzymatic responses to oestrogen. (S) Oestrogen added directly to the enzyme incubation medium did not change enzyme activities. (6) Weight loss in ovariectomized rats due to reduced food intake did not increase enzyme activities. (7) In the pituitary, good correlation was obtained between the known receptor binding properties of various oestrogenic and non-oestrogenic steroids and the elevation in G6PDH activity. The results indicate that oestradiol acts directly to cause changes in activities of some brain and pituitary enzymes. The possibility is discussed that these changes may result from oestrogen interaction with putative receptor sites found in pituitary and certain brain regions.     

The effects of n-3 polyunsaturated fatty acids by gavage on some metabolic enzymes of rat liver

In this experimental study, the effect of fish n-3 fatty acids was studied on the some important enzymes of carbohydrate metabolism, hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in rat liver. Wistar albino rats of experimental group (n= 9) were supplemented fish omega-3 fatty acids (n-3 PUFA) as 0.4 g/kg bw. by gavage for 30 days in addition to their normal diet. Isotonic solution was given to the control group (n= 8) by the same way. At 30th day, the rats were killed by decapitation under ether anesthesia, autopsied and liver was removed. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the liver. The n-3 PUFA caused increases in the activities of HK, G6PD, LDH, and MDH in comparison with control. These increases were statistically significant (P < 0.01) except 6PGD activity. As a result, n-3 PUFA may regulate the metabolic function of liver effectively by increasing HK, G6PD, 6PGD, LDH, and MDH enzyme activities of rat liver when added in enough amounts to the regular diet.     

Isolation of enzymes from mixed reversed micelles of surface-active agents

The catalytic activities of lysozyme, horseradish peroxidase (HP), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) were studied in aqueous solutions and after isolation of the enzymes from mixed reversed micelles of Aerosol OT and Triton X-45 by organic solvents (acetone, ethanol, isopropanol), by acetone-water mixtures, as well as by aqueous solutions containing urea, glycerol, polyethylene glycol 6000 and ammonium sulphate. The isolation conditions were found for catalase with retaining all the activity and for HP and lysozyme with retaining 72 and 84% of the catalytic activity, respectively. The G6PDH isolation from micelles by aqueous solutions of urea (6%) and glycerol (10%) resulted in retaining only 43% of the enzyme activity and led to almost complete inactivation of LDH. Stability of the enzymes after their entrapment in micelles and isolation from those is compared with thermostability of the same enzymes in aqueous solutions.     

Glycolytic enzymes in the premolt field crab Paratelphusa hydrodromus (Milne-Edwards) (Crustacea)

The quantitative assay of hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glucose 6-phosphate dehydrogenase (G-6-PDH), glycerol 3-phosphate dehydrogenase (G-3 PDH) and lactate dehydrogenase (LDH) revealed that coxal muscles compared to hepatopancreas contained higher activities of all the enzymes investigated. It appears that the coxal muscles of the premolt field crab has carbohydrate-based fuel economy. The hepatopancreas is a rich source of lipid and very poor source of glycogen. The activity of G-6-PDH is moderately high in the hepatopancreas. It seems that in this lipogenic tissue conversion of G-6-P to triose phosphate occurs predominately via pentose-phosphate pathway thus generating NADPH for lipogenesis. The relative G-3PDH ad LDH activities in hepatopancreas and coxal muscles led us to believe that the reconversion of NAD from NADH in hepatopancreas nd muscle flexor is effected by glycerol 3-phosphate shuttle, whereas in muscle extensor it is achieved by both G-3PDH and LDH activities.     

Interspecific hybridization between human and mouse somatic cells: Enzyme and linkage studies

Human-mouse somatic cell hybrids have been isolated and examined for enzyme and chromosome constitution. The enzymes assayed were lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH), NADP-dependent malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucomutase (PGM), and several esterases. Coexpression of mouse and human genomes and formation of heteropolymeric enzymes were observed in seven different hybrid populations for the enzymes LDH, IDH, MDH, and G6PD. Evidence predicated on the absence of chromosomal rearrangements is provided for the lack of genetic linkage in the human genome for these four enzymes, as well as for thymidine kinase.Supported in part by NIH Grants GM 09966 and 1-F1-GM-39,399 from the Institute of General Medical Sciences and by NIH Training Grant HD-32.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Microfluorometric study of glucose-6-phosphate and malate dehydrogenases in histologically defined areas of the uterus in cyclic and pregnant ewes

Activities of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49; G6PDH) and malate dehydrogenase (E.C. 1.1.1.37; MDH) were determined fluorometrically in freeze-dried sections of the sheep uterus during the estrous cycle and pregnancy. Samples (0.2–0.3 μg) from the luminal epithelium, uterine glands, maternal caruncles, fetal cotyledons and intercotyledonary trophoblast were assayed in a small aliquot (5 μl) of the reaction medium under oil.Activity of G6PDH in the luminal epithelium, uterine glands and maternal caruncles did not change during the estrous cycle. Activity of MDH in the uterine glands did not change during the cycle, but in the luminal epithelium and maternal caruncles highest activities were found on day 9 and day 2 post-estrus, respectively.The enzyme activities in the fetal tissues were lower than in the maternal tissues. In all maternal tissues, MDH and G6PDH activities decreased during early pregnancy, but after implantation, the activities increased significantly. In fetal tissues G6PDH activity increased, whereas MDH activity decreased during the second half of gestation. These results suggest an increased rate of pentose shunt activity in both maternal and fetal tissues, and an increased rate of Krebs' cycle activity in the maternal but not in the fetal tissues.     

Histochemical observations on the occurrence of glycolytic and pentose phosphate cycle enzymes in the hepatopancreas and their possible relation to eyestalk factor(s) in the crab Scylla serrata (Forskal).

Histochemical studies were carried out on some of the glycolytic enzymes viz. phosphorylase, aldose, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and lactic dehydrogenase (LDH) and a key enzyme of the pentose phosphatase cycle, glucose-6-phosphate dehydrogenase (G-6-PDH), in the hepatopancreas of Scylla serrata (Forskal). 1. Weak activities of phosphorylase and aldolase and strong-activities of alpha-GPDH and LDH were noticed mainly in the brush border of the tubules and R-cell cytoplasm. A trace activity of G-6-PDH was noticed in the brush border. 2. Bilateral eyestalk removal results in inhibition of both phosphorylase and aldolase. However, enhanced activities of alpha-GPDH and LDH were noticeable 4 h after the operation. The G-6-PDH activity remained unaltered till 24 h. 3. Injection of eyestalk extract into both intact and destalked crabs activated all the enzymes.     

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

Summary The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media. These results clearly show that the gel method is not suitable for the valid demonstration of activity of (partially) soluble enzymes. Furthermore, our results confirm that a greater proportion of G6PDH than of LDH is present in a soluble form in liver cells.     

Relations of enzymes inAspergillus repens grown under sodium chloride stress

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2m sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2m NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na⁺ (as NaCl) when added up to 100mm in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.     

The histochemistry of developing adipocytes in primary stromal-vascular cultures of rat adipose tissue

Summary Histochemical analysis for NADP-dependent dehydrogenases, succinate dehydrogenase, NADH and NADPH-tetrazoleum reductases and esterase was conducted on primary cultures of adipose tissue stromal-vascular cells. Enzyme activities were restricted to clusters of lipid laden cells (adipocytes). The number of enzyme reactive adipocytes increased with length of culture. Coverslips were partially coated with collagen to allow comparisons of cell differentiation on coated (C-glass) and uncoated glass (U-glass) surface. There were no reactions for NADH- and NADPH-tetrazoleum reductases (TR) in cells on C-glass whereas adipocytes and stromal cells on U-glass were reactive. Glucose-6-phosphate (G6PDH) and 6-phosphogluconate (6PGDH) dehydrogenase activities were markedly demonstrated in both stromal cells and adipocytes on U-glass. Malate (MDH) and isocitrate (ICDH) dehydrogenase activites were higher in adipocytes than in stromal cells on the U-glass. Stromal cells on C-glass were either devoid of these enzymes (G6PDH, MDH, 6PGDH, ICDH) or activity was restricted to a small area of the cytoplasm. There were two levels of staining intensity in (MDH, ICDH, G6PDH, 6PGDH) adipocyte clusters on C-glass.Elimination of phenazine methosulphate from the NADP-dependent dehydrogeanse medias and SDH media, caused a reduction in enzyme reactive adipocytes on the C-glass. This manipulation did not reduce the number of enzyme reactive cells on U-glass. Cells on C-glass and U-glass were distinctly different in esterase stained coverslips. These studies demonstrated enzyme histochemical reactions of adipocytes and stromal cells in primary culture that were dependent on the type of extracellular matrix. Furthermore, enzyme histochemistry was shown to be useful for delineating adipocytes from stromal cells in primary cultures.     

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.