Kenneth L. Barker

Glucose-6-phosphate dehydrogenase. Translational regulation of synthesis and regulation of processing of the enzyme in the uterus by estradiol

Abstract 12 h after intravenous administration of estradiol to ovariectomized mature rats, and 18-fold increase in the rate of synthesis of glucose-6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADP + oxidoreductase, EC 1.1.1.49) is observed. At that time, functional mRNA pools coding for this protein are only elevated 7-fold, suggesting that estradiol may be exerting effects at a posttranscriptional level of protein synthesis. The effect of estradiol on the rate of translation of the mRNA for this enzyme was evaluated by estimating the average ribosomal transit times in control and estradiol-treated rats under in vivo conditions. Transit times for glucose-6-phosphate dehydrogenase were decreased from 4.9 min in estrogen-deprived rats to 2.0 and 3.4 min, respectively, in 12 or 24 h estradiol-treated rats. Transit times for total released proteins in the cytosol were similarly decreased from 2.3 min to 1.0 and 1.3 min in the same groups of animals. Glucose-6-phosphate dehydrogenase has pyroglutamate as its NH 2 -terminus indicating that this region of the protein exists in a transient precursor form. The difference between the expected time after [ 3 H]glutamate administration that [ 3 H]pyroglutamate should be found in the enzyme and the observed first occurrence of [ 3 H]pyroglutamate in the enzyme is 9, 2.5 and 4 min in control and 12 or 24 h in estradiol-treated rats, respectively. This indicates that the ‘processing’ or removal of an NH 2 -terminal peptide from a percursor form of this enzyme is finalized after release of a glucose-6-phosphate dehydrogenase precursor from ribosomes and that the processing event is enhanced by estradiol.

Competitive effects of estradiol-17α on the estradiol-17β induced physical changes in uterine dna

Abstract The presence of estradiol-17β during the melting of native human uterine DNA results in a hyperchromic shift with a Tm of about 35°C. The shift is not observed in the absence of estradiol-17β. The degree of the shift (A 260 at 40°C - A 260 at 25°C) is proportional to the amount of estradiol-17β in the melting mixture up to 4 × 10 −5 M estradiol-17β. Addition of equimolar concentration of estradiol-17α completely abolishes the 35°C shift. The hyperchromicity of DNA is exactly proportional to the concentration difference [(E 2 -17β) - (E 2 -17α)] of the two steroids and equals the shift caused by an amount of estradiol-17β equivalent to the difference. Equilibrium dialysis indicates that small amounts of 3 H-estradiol-17β are bound to uterine DNA and that this binding is abolished by addition of equimolar amounts of estradiol-17α. The sequence of addition of the two steroids did not affect competition by estradiol-17α. The estradiol-17β induced hyperchromicity at 35°C was observed to occur in DNA prepared from both lung and uterine tissue of rats. The unusual type of competition between estradiol-17β and estradiol-17α in this response, suggests that instead of competing for DNA binding sites, estradiol-17α might bind to or titrate estradiol-17β, making it inactive.

Glucose-6-phosphate dehydrogenase Partial characterization of the rat liver and uterine enzymes

Some properties of rat liver and uterine glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) have been determined. A procedure has been used for the purification of rat liver glucose-6-phosphate dehydrogenase to homogeneity (spec. act. 210-225 units/mg protein) from large amounts of liver (0.5-2 kg) with yields of up to 30%. Uterine glucose-6-phosphate dehydrogenase was obtained by immunoprecipitation methods and the properties of radioactively-labeled forms of this enzyme were then determined. The amino acid composition of the liver enzyme was found to be similar to that for the enzyme from other mammalian tissues. The liver and uterine enzymes have a subunit molecular weight of 57000 and a pI of 6.5. The NH2-terminal amino acid of both enzymes was found to be pyroglutamate.

Regulation of the peptide elongation reaction on uterine ribosomes by estrogens

Administration of 17 beta-estradiol to ovariectomized mature rats for 1 h induces an increased capacity of subsequently isolated uterine ribosomes to synthesize protein in a cell-free protein synthesis system. The increased rate of protein synthesis can be ascribed to an effect of estrogen on the rate of peptide elongation rather than synthesis of additional new peptides. The increased rate of peptide elongation is dependent upon the dose of estradiol over the range of 0.1 to 10 micrograms/animal, and exhibits hormone specificity; 17 beta-estradiol, diethylstilbesterol, estrone and estriol but not 17 alpha-estradiol, progesterone, dihydrotestosterone or corticosterone will induce the response. Removal of ribosome associated proteins by extraction with 0.5 M KCl results in activation of protein synthesis by uterine ribosomes from control rats to rates that are equal to that of ribosomes from estrogen-stimulated rats suggesting that ribosomes from control animals are in an inhibited state. The KCI extracted ribosomal factors from control animals inhibit the synthesis of protein by salt-washed uterine ribosomes when added back to the ribosomes prior to assay and the inhibitory properties of these factors are greater if derived from ribosomes of control rather than 1 h estradiol-treated rats. The extracted inhibitor is inactivated by heat, is insensitive to treatment with N-ethylmaleimide, is insensitive to micrococcal nuclease and is reversible. The early activation of uterine ribosomes by estrogen appears to result from either the removal or inactivation of a ribosome associated-peptide elongation reaction, inhibitory factor.

Regulation of the Levels of mRNA for Glucose-6-phosphate Dehydrogenase and Its Rate of Translation in the Uterus by Estradiol

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyzes the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconolactone with the simultaneous reduction of NADP+ to NADPH. The enzyme is the first in the pentose phosphate pathway (hexose monophosphate shunt), which produces pentose phosphates used for nucleotide and nucleic acid synthesis, and reducing equivalents in the form of NADPH for use in biosynthetic reduction reactions, including fatty acid synthesis and hydroxlations. G6PD has been purified and characterized from the tissue cytosols of several mammalian species, including human erythrocytes, rat mammary gland, rat and mouse liver, and bovine adrenal cortex (Bonsignore and DeFlora, 1972; Levy, 1979). In general, the enzymes from these sources are “similar” and are composed of subunits that, by immunological and molecular weight criteria, may be identical proteins within a species. The monomeric subunits have a molecular weight of about 60,000, based on their rate of migration on SDS-polyacrylamide gels. The monomers are catalytically inactive. In the presence of NADP+, the inactive monomers form a catalytically active dimer containing 1 or perhaps 2 moles of NADP+ per mole of dimer; at reduced ionic strength and pH, and in the presence of Mg2+, the dimers aggregate to form tetramers and hexamers.

Effect of sodium fluoride on glucose-6-phosphate dehydrogenase activity in the rat uterus.

Intrauterine administration of 50 mumol of NaF to the ovariectomized mature rat causes a 2--3-fold increase in the total uterine glucose-6-phosphate dehydrogenase activity within 24 h. The response is characterized by a 4--6 h lag with a maximum effect from 24 to 36 h after a single treatment. Uterine glucose-6-phosphate dehydrogenase activity continues to increase with daily administration of NaF through 4 days. The NaF-induced response is blocked by prior intrauterine administration of cycloheximide but not actinomycin D suggesting that the enzyme activity increases by a post-transcriptional effect of NaF on de novo enzyme synthesis. Direct measurement of the effect of NaF on the rate of incorporation of [14C] leucine into immunoprecipitable uterine glucose-6-phosphate dehydrogenase indicates that NaF causes a 9-fold increase in the rate of enzyme synthesis during the interval from 12 to 16 h after treatment. The half-life of the enzyme as measured by the rate of loss of [1-14C] glutamate from previously labeled utreine glucose-6-phosphate dehydrogenase is decreased from 27 to 10 h by NaF. The NaF response does not seem to be mediated by activation of uterine adenylyl cyclase since theophylline does not potentiate the response and since intrauterine application of cyclic AMP does not mimic the response. The increase in enzyme activity is preceded by an increase in the rate of utilization of the hexose monophosphate shunt pathway as determined by the ratio of the the rates of oxidation of [1-14C]glucose to [6-14C] glucose to CO2 by uterine slices in vitro. The action of NaF on this pathway most likely resutls from inhibition of the glycolytic enzyme, enolase, and increased pathway utilization may be the factor which controls enzyme synthesis. When given in combination with other known inducers of uterine glucose-6-phosphate dehydrogenase such as estradiol and NADP+, NaF acts synergistically.

Effect of Stage of Development and Function on the Template Activity of Rat Mammary Gland Chromatin

Summary The ability of purified mammary gland chromatin to serve as a template for DNA-dependent RNA polymerase increases slightly during gestation and increases nearly twofold after the onset of lactation. The composition of the purified chromatin changes at parturition and is characterized by a 2-3-fold increase in RNA, a 1.2-1.3-fold increase in histone, amd a 1.1-1.7-fold increase in nonhistone chromatin proteins. Since the synthesis of milk proteins begins coincidentally with the increase in chromatin template activity, the response probably results from the expression of a new set of genes although the possibility of an increase in the rate of expression of previously active genes cannot be excluded.

The Role of Modern Biology and Medicine in Drug Development in Academia and Industry

This symposium addresses careers in drug development in industry; the performance of translational research by academia, industry, and both; and numerous factors pertinent to alliances essential to drug discovery and development. Drug development is a complex process that regularly involves effective collaborations between academic and physician scientists and industry. There are specific occupational factors affecting recruitment of scientists and physicians in drug development programs in industry; ideal backgrounds for successful applicants for positions in industry in drug development; ethical and regulatory considerations particularly germane to the performance of scientists and physicians in drug development programs in industry and at universities; and particular gratifications available to scientists in Industry working on drug development. Both similarities and differences characterize the performance of translational research in industry compared with academia. In industry, logistic, operational, and scientific oversight is complex, especially because It often Involves relationships with clinical enterprises outside of the corporation. The process is long and arduous from formulation of a good Idea in discovery to acceptance of a novel drug in the marketplace. Collaborations and partnerships by industry often involving academia and confrontation of multiple Issues are pivotal.

Effects of 8-Azaguanine on the Induction of Uterine Glucoses-Phosphate Dehydrogenase Activity by Estradiol or NADP+

Summary The purine analogue, 8-azaguanine, inhibits the induction of uterine glucose-6-P dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) by estradiol or the enzymes co-factor, NADP+. Inactivation of the enzyme after estradiol withdrawal was not affected by 8-azaguanine treatment. Since 8-aza-guanine limited the uterine response to estradiol when injected at any time after the steroid, the RNA necessary for the synthesis of uterine glucose-6-P dehydrogenase must be synthesized continuously after the initiation of the uterine response to estradiol. This research was supported by USPHS Research Grant (HD-02,851), National Institute of Child Health and Human Development.