Catalytic activity of conjugates of peroxidase with progesterone in aqueous and micellar media in the presence of antibodies against progesterone

At 22 and 41 degrees C the horseradish peroxidase (HRP) conjugates with progesterone, HRP-PROG-1 and HRP-PROG-2, were obtained by HRP reaction with N-hydroxysuccinimide ester of 3-O-carboxymethyloxime of progesterone. The interaction of these conjugates and unmodified enzyme with antibodies against progesterone was studied in phosphate-citrate buffer pH 3.6 and in the reversed micelles of Aerosol OT and Triton X-45 in heptane. Catalytic function of conjugates in buffered solutions and in reversed micelles increases as result of the conjugates' interaction with antibodies. The free progesterone in reversed micelles competes with HRP-PROG-1 for antibody binding, which may be the basis for homogeneous enzyme-immunoassay of progesterone, depending on the micelle composition, the antibody concentration and temperature.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

Catalytic activity of steroid conjugates of various types of peroxidases in aqueous and micellar media

The horseradish peroxidase (HRP) conjugates with the sheep antirabbit antibodies and cortisol (COR) or progesterone (PROG) containing 9 to 40 steroid molecules per HRP molecule were synthesized. In aqueous media all the conjugates have lower catalytic activity in the o-phenylenediamine oxidation than the native enzyme. In reversed Aerosol OT micelles in heptane the HRP-COR and HRP-PROG conjugates containing 12 and 9 steroid molecules, respectively, have catalytic constants 2.6 and 2.7 times higher than the unmodified enzyme. The influence of the HRP hydrophobisation and its inactivation on the course of modification on catalytic properties of the enzyme is discussed.     

Catalytic activity and thermostability of dehydrogenase conjugates with cortisol and progesterone.

The conjugates of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase with progesterone and cortisol, containing 1-40 steroid molecules per enzyme molecule, were obtained by the reactions of N-succinimide esters of the 3-[O-(carboxymethyl)oximes)] of cortisol and progesterone with a protein in a water-DMFA (10%) medium. The catalytic activity and thermostability of dehydrogenases and their steroid conjugates were kinetically studied. The effects of the modification degree on the activity and thermostability of dehydrogenases by their hydrophobization were studied and discussed. Practical recommendations for using the dehydrogenase-steroid conjugates in enzyme immunoassay are given.     

Erythrocyte glucose-6-phosphate dehydrogenase activity in Brazilian monkeys

Activities of glucose-6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase as well electrophoretic mobility of glucose-6-phosphate dehydrogenase from erythrocytes of Brazilian monkeys were investigated. Glucose-6-phosphate dehydrogenase activity of simian was 4 times higher than the human values. Regarding electrophoretic studies, the results, did not reveal any intraspecific polymorphism. A comparison of erythrocyte glucose-6-phosphate dehydrogenases among primates is also presented.     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in livers of Ni-deficient rats.

In experiments using rats it was shown that inadequate dietary supply of Ni reduces growth and lowers the erythrocyte count, hematocrit and hemoglobin level in blood, that the Ni supply affects the trace element content of iron, copper and zinc in various body organs, and that the absorption of iron is greatly impaired by Ni deficiency. For further biochemical criteria on the essentiality of nickel, the activities of two dehydrogenases, malate dehydrogenase and glucose-6-phosphate dehydrogenase, were measured in liver homogenates from two generations of rats at 30 and 50 days of age. In the 30-day-old rats of both the F1 and F2 generation, the activity of the malate dehydrogenase fell to about two-thirds the level of control animals. In the liver of the 50-day-old rats the activity of this enzyme was about the same in deficient animals as in the controls. The activity of glucose-6-phosphate dehydrogenase of Ni-deficient rats was reduced by 85% in the F1 generation and by 56% in the F2 generation at 30 days of age as compared with control levels. In 50-day-old rats the activity had fallen to half the level of control animals at 30 days of age. At the age of 50 days, there was no significant difference between the deficient and the control groups of either generation.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Ultrastructural demonstration of glucose-6-phosphate dehydrogenase activity in steroid-secreting cells

Summary The ultrastructural localization of glucose-6-phosphate dehydrogenase (NADP-linked) has been attempted in steroid-secreting cells. Rat adrenocortical cells and newt testicular glandular cells were fixed in an ice-cold mixture of 1% methanol-free formaldehyde and 0.25% glutaraldehyde. Potassium ferricyanide was used as the final electron acceptor.After incubation, the final copper ferrocyanide precipitate is exclusively observed in the hyaloplasm of these cells, provided that an electron carrier (1.0 mM PMS) has been added to the medium in order to by-pass the tissue diaphorase (NADPH-ferricyanide reductase) reaction. No precipitate appears in the absence of glucose-6-phosphate (substrate). Incubation in a medium devoid of PMS results in an exclusively mitochondrial reaction; the latter is that of the diaphorase, which in these cells is mitochondrial. These results prove the importance of utilizing exogenous electron carriers (such as PMS) in coenzyme-linked dehydrogenase cytochemistry.Although polyvinyl alcohol was included in the washing and incubation media, in order to increase their viscosity, problems still exist concerning ultracytochemical localization of this soluble enzyme; these problems are discussed in the paper.     

Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

The effect of hydrophobization of glucose-6-phosphate dehydrogenase with progesterone on its thermal inactivation

Thermal inactivation of glucose-6-phosphate dehydrogenase (G6PDH) and its conjugates with progesterone containing 3, 7 and 35 molecules of the modifier was studied in bidistilled water over a temperature range 35-47 degrees. At different temperatures and initial concentrations of the enzyme and its modified forms, thermal inactivation is described by the equation of the first order up to a significant degree of enzyme deactivation. The effective Kin values are decreased with the increase of the native G6PDH concentration and changed in a complicated manner with the increase of the conjugate concentration depending on the enzyme modification degree, which reflects a great role of the enzyme hydrophobicity in its inactivation. The role of hydrophobicity of the modified G6PDH in changes of its specific activity is discussed.     

Enhanced proteolysis of glucose-6-phosphate dehydrogenase in the presence of palmitoyl coenzyme A

Palmitoyl coenzyme A at concentrations below its critical micelle concentration increases the rate of proteolysis of baker's yeast glucose-6-phosphate dehydrogenase by proteinase A in the pH range 4-5. both glucose-6-phosphate and NADP protect glucose-6-phosphate dehydrogenase against proteolysis, but these protective effects are diminished in the presence of palmitoyl coenzyme A. Since palmitoyl coenzyme A is known to dissociate glucose-6-phosphate dehydrogenase into dimers, the results imply that the in vivo half life of glucose-6-phosphate dehydrogenase may be controlled by a process based on the regulation of the oligomeric structure of the enzyme by the collective actions of various molecules, including palmitoyl coenzyme A.