The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.     

Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes

Both normal and chronic granulomatous disease (CGD) neutrophils were able to degrade the subendothelial matrix secreted by human endothelial cells via an elastase-dependent process. In the absence of the plasma antiproteinase, alpha-1-proteinase inhibitor (alpha-1-PI), normal neutrophils protect their released elastase from inactivation by using the chlorinated oxidants hypochlorous acid and endogenous N-chloroamines to suppress the antiproteinase's activity. In contrast, CGD neutrophils were unable to generate either class of chlorinated oxidant or to inactivate the porcine pancreatic elastase inhibitory capacity of alpha-1-PI unless the cells were supplemented with exogenous hydrogen peroxide. Despite the reliance of normal neutrophils on chlorinated oxidants to inactivate alpha-1-PI, neutrophils triggered in the presence of agents that block the generation of these reactive species continued to degrade the subendothelial matrix at a suppressed but significant rate in the presence of a 50-fold excess of the antiproteinase. The continued solubilization of the matrix by normal neutrophils was not due to the incomplete inhibition of oxidant generation because triggered CGD neutrophils were also able to degrade the matrix in the presence of excess alpha-1-PI. If CGD neutrophils were stimulated in the presence of an exogenous source of H2O2 and alpha-1-PI, the proteolytic potential of the cells was identical to that observed with normal stimulated neutrophils. We conclude that normal neutrophils can enhance their ability to degrade the subendothelial matrix by oxidatively protecting elastase from inactivation by alpha-1-PI but both normal and CGD neutrophils possess non-oxidatively linked mechanisms for sequestering and using elastase to mediate proteolytic effects in the presence of native antiproteinase.     

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).     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase of human malignant cells by methylglyoxal

The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Inactivation of alpha-1-proteinase inhibitor in serum by stimulated human polymorphonuclear leucocytes. Evidence for a myeloperoxidase-dependent mechanism

Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha-1-proteinase inhibitor (alpha-1-PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha-1-PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha-1-PI in the presence of exogenously-added PMNL-derived supernatants which contain this enzyme. Cell-free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6.2, the pH optimum for myeloperoxidase-catalysed inactivation of alpha-1-PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha-1-PI by generating hypochlorous acid in the presence of chloride and respiratory burst-derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactivate the most important antielastase, alpha-1-PI.     

Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities.

Oocytes at several stages of growth were isolated by enzymatic digestion of ovaries from infant mice. These oocytes were free of follicle cells and were obtained in sufficient numbers to permit direct biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) activities. Both enzymes increase in total activity as the oocyte grows. However, while the specific activity of G6PD remains constant up to an oocyte diameter of 80–85 μm, the specific activity of LDH increases four-fold. The specific activity of G6PD and especially LDH declines in oocytes over 80–85 μm in diameter, suggesting that the synthesis of these enzymes is almost completed at this stage of growth.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

Uric acid substantially enhances the free radical-induced inactivation of alcohol dehydrogenase

Lactate dehydrogenase (LDH) and yeast alcohol dehydrogenase ( YADH ) are inactivated when attacked by hydroxy free radicals (OH). Organic molecules with a high rate constant of reaction with OH such as ascorbate or urate can compete with the enzymes for these strongly oxidising radicals. However, although 10(-3)M ascorbate can substantially protect both LDH and YADH from OH attack, in the presence of 10(-3)M urate only LDH is protected. In the case of YADH an even greater degree of inactivation than with OH occurs. The extent of inactivation is considerably reduced when oxygen is absent, in agreement with a urate peroxy radical perhaps being partly responsible for the increased inactivation of the enzyme.     

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.     

Effects of diallyl sulfide and zinc on testicular steroidogenesis in cadmium-treated male rats

Cadmium (Cd) is one of the environmental pollutants that affect various tissues and organs including testis. Harmful effect of cadmium on testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. In the present study, the effect of diallyl sulfide (DAS), a sulfur-containing volatile compound present in garlic, and zinc (Zn) was investigated on cadmium-induced testicular toxicity in rats. Male adult Wistar rats treated with cadmium (2.5 mg/kg body wt, five times a week for 4 weeks) showed decreased body weight, paired testicular weight, relative testicular weight, serum testosterone, luteinizing hormone, follicle-stimulating hormone, and testicular total antioxidant capacity (TAC) and protein levels. Testicular steroidogenic enzymes, such as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and marker enzymes, such as sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP), and glucose-6-phosphate dehydrogenase (G6PD), showed a significant decrease in activities whereas that of gamma-glutamyl transferase was significantly increased after cadmium exposure. The results have revealed that concurrent treatment with DAS or zinc restored key steroidogenic enzymes, SDH, LDH, and G6PD and increased testicular weight significantly. DAS restored the TAC level and increased testosterone level and relative testicular weight significantly. Zinc restored testicular protein level and body weight. It can be concluded that cadmium causes testicular toxicity and inhibits androgen production in adult male rats probably by affecting pituitary gonadotrophins and that concurrent administration of DAS or zinc provides protection against cadmium-induced testicular toxicity.     

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.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.     

Human alpha-1-antichymotrypsin: purification and properties.

Human alpha-1-antichymotrypsin has been purified to homogeneity by the following sequential steps--(a) ammonium sulfate fractionation; (b) chromatography on Cibacron Blue Sepharose at pH 7.0; and (c) chromatography on SP-Sephadex C-50 at pH 5.5. The inhibitor has a molecular weight near 68,000 and contains approximately 26% carbohydrate alpha-1-Antichymotrypsin has an amino-terminal arginine and a carboxy-terminal glycine. It also has some homology with alpha-1-PI based on amino-terminal sequence analysis of both proteins. Complexes of alpha-1-antichymotrypsin with human chymotrypsin and human leukocyte cathepsin G are stable in sodium dodecyl sulfate and have molecular weights near 90,000 suggesting 1:1 complex formation on a molar basis between inhibitor and enzyme.     

Red cell enzymes of primates (Anthropoidea)

The patterns of five red cell enzymes (acid phosphatase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, adenylate kinase, and phosphoglucomutase) in various Anthropoidea were compared on starch gels. Small samples of gorilla, orang, and gibbon, six genera of Old World monkeys, and seven genera of New World monkeys were examined. Among the hominoids the gibbon was the most divergent. The langur differed markedly from macaques and baboons in certain enzyme patterns. Most of the ceboids had relatively high G6PD activities. Individual variations in 6PGD, AP, AK, and PGM but not in G6PD were seen in some species. Many genera could be distinguished by the mobility of the achromatic bands.     

Effects of controlled exposure of L cells to bromodeoxyuridine: II. Turnover rates and activity profiles during cell cycle of bromodeoxyuridine-sensitive and -resistant enzymes

Fluorodeoxyuridine (FUdR)-synchronized mouse L cells were allowed to incorporate 5-bromodeoxyuridine (BUdR) at restricted intervals in the S phase and the effects of the selective incorporation of BUdR in DNA on the activities of seven randomly chosen enzymes (five dehydrogenases and two phosphatases) were analysed. Reductions to 56.9 and 83.3 % of the control levels were noted for glucose-6-phosphate dehydrogenase (G6PD) and alcohol dehydrogenase (ADH) activities respectively, when cells were exposed to BUdR during the 1st h of S. Acid phosphatase (AcP) activity was reduced to 81.9% of the control level following exposure to the analogue during the 3rd h of S. Exposure of cells to BUdR for the entire S period failed to increase the magnitude of the reductions in activity for any of these three enzymes. Alternately, when cells were allowed to synthesize DNA in the presence of thymidine for the 1st h of S and the remainder in the presence of BUdR, the activities of G6PD and ADH were comparable to those found in untreated cells. Exposure of cells to thymidine for the 3rd h of S, combined with exposure to BUdR for the preceding and subsequent hours of S, provided complete protection against the BUdR-mediated reduction in AcP activity. The activities of lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6pGD), isocitrate dehydrogenase (IDH) and alkaline phosphatase (A1P) were found to be insensitive to treatment with BUdR, even when the period of analogue exposure encompassed the entire S period.Additional investigations carried out with G6PD for characterization of the nature of the BUdR effects suggest that the BUdR-mediated reductions in enzyme activities are not caused by the increased rates of degradation of the enzymes, formation of enzyme inhibitors or by the disproportionate replication of A-T base pairs during BUdR treatment. The alterations of enzyme activities appear to result from decreased rates of synthesis of enzymes in BUdR-treated cells. The results of the present study clearly suggest that pulse labelling of cells with BUdR at various intervals of the S phase may provide a useful approach for determining temporal localization of replication time of DNA segments that are critical for the synthesis or regulation of specific gene products.     

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.     

Effect of a polyherbal formulation, Ambrex, on butylated hydroxy toluene (BHT) induced toxicity in rats

Effect of polyherbal formulation Ambrex was evaluated in butylated hydroxytoluene (BHT) induced toxicity of lungs and liver in rats. Toxicity was produced by administering BHT (500 mg/kg/day) for 3 days. Lung damage was evidenced by elevated levels of broncho alveolar lavage fluid (BAL) parameters such as protein, lactate, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and glucose-6-phosphate dehydrogenase (G6PDH). Liver damage was proved by elevated levels of serum protein and markers such as LDH, ALP, aspartate amino transferase (AST), alanine amino transferase (ALT), decreased level of lipid peroxides (LPO) in serum and glutathione (GSH) in liver. Administration of aqueous suspension of Ambrex (50 mg/kg orally) retained these elevated levels of BAL-protein, lactate, LDH, ALP, ACP, G6PDH and serum-protein, LDH, ALP, AST and ALT at near normal values. Decreased level of liver GSH was retained at near normalcy in Ambrex pretreated BHT-administered animals. There was no change in liver LPO in all the four groups.