Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase

A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 3–4 m, as well as from the hydroxylapatite column at a phosphate concentration of 2–3 m.     

Hydrolysis by horse muscle acylphosphatase of (Ca2+ + Mg2+)-ATPase phosphorylated intermediate

Horse muscle acylphosphatase (EC 3.6.1.7) was found to hydrolyze the labeled phosphorylated intermediate of (Ca²⁺ + Mg²⁺)-ATPase from rabbit muscle. In addition, the phosphorylated peptides obtained by pepsin digestion of the labeled phosphorylated microsomes were completely hydrolyzed by acylphosphatase. These findings suggest a possible regulatory role of this enzyme in vivo on the calcium transport process by sarcoplasmic reticulum.     

Hydroxo-complexes of Hg2+ chelates as models of hydrolytic metalloenzymes

Hydroxo-complexes of chelates of Hg²⁺ were found to catalyze hydrolysis of active esters and amides. Thus, 10⁻²M of the hydroxo-complex of Hg²⁺ pentamethyl diethylenetriamine (Hg-PDETA) at pH 7 enhanced the rate of hydrolysis of p-nitrophenyl esters and of cinnamoyl imidazole by factors of 400 and 1900, respectively. In the latter case the rates of reaction were linear with catalyst concentration. The hydroxo-complexes of Hg²⁺ phenanthroline (Hg-Phen) exhibited kinetic specificity toward p-nitrophenyl carbalkoxyglycinates. With these specific substrates acceleration factors of 1000 and more were obtained at 5 × 10⁻³M Hg-Phen, pH 8. The dependence of rates upon catalyst concentration was found to be curvilinear. This latter behavior was attributed to attack of one molecule of Hg-Phen, in the form of a hydroxo-complex, on a ternary complex Hg-Phen-substrate. The general features of metal-ion-catalyzed hydrolytic reactions are discussed and compared with the mode of action of hydrolytic metalloenzymes such as carboxypeptidase A and carbonic anhydrase.     

Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.     

Induction of fructose 1,6-bisphosphatase and glucose 6-phosphatase in fetal mouse liver

Fructose 1,6-bisphosphatase and glucose 6-phosphatase were induced in organ cultures of liver tissues from 15- and 19-day-old fetal mice, using a culture method that allowed the tissues to be maintained for 7 days in the absence of serum. In cultures from 15-day-old fetal liver, both enzyme activities increased significantly per milligram of DNA after a lag period of 1 to 3 days. In cultures from 19-day-old fetal liver only glucose 6-phosphatase increased in the absence of inducer. N6,O2'-Dibutyryladenosine 3',5'-monophosphate enhanced the rate of increase in fructose 1,6-bisphosphatase and glucose 6-phosphatase activities. The minimum effective concentration of the cyclic nucleotide was approximately 10(-6) M. Dexamethazone inhibited the increase in fructose 1,6-bisphosphatase during culture for 7 days. Glucose 6-phosphatase activity was enhanced by dexamethazone in cultures from 19-day-old fetal liver, but was without effect on glucose 6-phosphatase in cultures from 15-day-old fetal liver. The minimum inhibitory concentration of dexamethazone was less than 10(-8) M. The results suggest a complicated effect of the cyclic nucleotide on the two enzymes in fetal mouse liver as well as different mechanisms of action of dexamethazone on the induction of two enzymes.     

Regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones

The regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones has been studied following the administration of 3,3',5-triiodo-L-thyronine and/or triamcinolone to hypophysectomized rats. The apparent Km for glucose-6-P in isolated ("intact") microsomes increased following administration of either hormone; there was little or no difference in the apparent Km when microsomes were treated with sodium deoxycholate ("disrupted"). In intact microsomes, triiodothyronine caused a 2.3-fold increase in the Vmax of glucose 6-phosphatase; triamcinolone, a 4-fold increase; and both hormones together, a 4.4-fold increase. Corresponding values for disrupted microsomes were: triiodothyronine, 3.7-fold; triamcinolone, 1.8-fold; both hormones, 3.3-fold. After triiodothyronine treatment, disruption of microsomes caused an over 5-fold increase in Vmax; after triamcinolone treatment, the increase was only 1.5-fold. This difference could not be explained by a change in the energy of activation of glucose 6-phosphatase in either intact or disrupted microsomes following hormone treatment. Glucose 6-phosphatase was localized by a cytochemical procedure; the reaction product was associated with 90% of the profiles in all microsomal preparations, except for those from triiodothyronine-treated rats, where less than 50% contained lead precipitate. Vesicles free of lead phosphate were isolated from sucrose gradients and accounted for less than 10% of the protein and glucose 6-phosphatase in all preparations, again except for those from triiodothyronine-treated rats, where they represented 40% of both the protein and glucose 6-phosphatase. The results are consistent with a model for glucose 6-phosphatase in which the substrate is transported across the microsomal membrane by a specific carrier before hydrolysis within the cisternae by a phosphohydrolase. It is suggested that the effect of triiodothyronine is mainly on the activity of the phosphohydrolase, and triamcinolone, on that of the carrier.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.     

Flow-through viscometer for use in the automated determination of hydrolytic enzyme activities: Application in protease,amylase, and pectinase assays

A flow-through viscometer is described, developed for application as a sensor in automated analysis. Its essential part is a glass capillary connected to the sample flow circuit with thin-walled rubber tubes at both ends. These tubes separate the fluid to be tested from a hydraulic liquid. This construction ensures the absence of dead space and a minimal test volume The usefulness of the apparatus is demonstrated in the automated assay of protease, amylase, and pectinase activity. Development of a mathematical model describing the enzymic degradation of macromolecules resulted in a reciprocal equation allowing rectilinear presentation of the calibration data. The feasibility of this model was tested by linear regression analysis of the data.     

Location of Mn2+ in concanavalin A containing only a Mn2+ ion

The metal-sugar distances in two metallized forms of concanavalin A have been compared by ¹⁹F magnetic resonance techniques. Using relaxation times measured at two different frequencies we have shown that the distance between the Mn²⁺ ion and the bound sugar in concanavalin A containing only Mn²⁺ is essentially identical to that found in concanavalin A containing both Mn²⁺ and Ca²⁺. Our results rule out the possibility that Mn²⁺ activates concanavalin A by binding at the Ca²⁺ site (S2) and would suggest that Mn²⁺ alone can induce an active saccharide binding conformation by binding at the transition metal site (S1).     

Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

The differential effects of sulfhydryl (SH)-blocking agents on hormone and DNA binding by the chick oviduct progesterone receptor were investigated. Previous studies have demonstrated inhibition of steroid-receptor interaction by SH-blocking agents and protection against inhibition by bound hormone. The present results indicate that the SH group required for steroid binding is within or near the hormone-binding site itself, and that a second SH group (or groups) is involved in the binding of receptor to DNA. Three findings relate to the site of action of SH-blocking agents on hormone binding. First, glycerol decreased the rate of hormone dissociation and the rate of hormone displacement by mercurial reagents by 75 to 90%. Second, mercurial reagents displaced [³H]progesterone bound to the mero-receptor, a M_r 23,000 proteolytic fragment containing the hormone-binding site, but not the site of interaction with DNA. Third, hormone displacement was still present after a 10,000-fold purification of the progesterone receptor. Mercurial reagents also inhibited binding of progesterone receptor to DNA, whereas the SH-alkylating agents N-ethylmaleimide and iodoacetamide had no effect. It is likely that distinct sulfhydryl groups are required for steroid receptor interaction with hormone and with DNA, since brief treatment with mercurial reagents blocked DNA binding, but caused only a slight displacement of bound hormone. The SH group required for hormone binding probably lies within or near the hormone-binding site, is sensitive to mercurials, alkylating agents, and 5,5′-dithiobis(2-nitrobenzoate) (DTNB), and is protected by bound hormone. The SH group required for DNA binding, in contrast, is sensitive to mercurials but not to alkylating agents, is only partially sensitive to DTNB, and is not protected by bound hormone.     

A new synthetic route to 2-hydroxyl steroidal estrogens via acetylation and dakin oxidation

This paper describes a new two-step synthetic route to 2-hydroxy estrogens from either estrone or estradiol, via 2-acetylation followed by Dakin oxidation. This approach is characterized by its simplicity and excellence of yield.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Binding of Ca2+ and Mg2+ to phosphatidylserine vesicles: different effects on P-31 NMR shifts and relaxation times.

Phosphorus-31 NMR lines corresponding to inner and outer surfaces of sonicated phosphatidylserine vesicles can be distinguished by the effects of added Ca²⁺ or Mg²⁺ at low bulk concentrations (millimolar or less). The changes in chemical shift and relaxation times indicate that Ca²⁺ binds directly to the PS phosphate, neutralizing at least a portion of the negative charge and restricting the motion of this group. Mg²⁺ ion also binds to the head group, but apparently not as strongly as Ca²⁺, nor is the mobility of the headgroup affected as much.     

Stereochemical aspects of synthetic and naturally occurring 2-hydroxy fatty acids. Their absolute configurations and assays of optical purity

d- and l-Cerebronic acid⁵, ⁶ methyl esters were obtained from a dl-mixture by converting the racemic ester to the l-acetylmandelates and separating the diastereomers by thin-layer chromatography; the diasteromers were then decomposed with mild acid methanolysis. The optical purities of the d- and l-enantiomers were determined as described below and found to be 82.5% and 87.1%, respectively. Absolute configurations of the enantiomers were confirmed by their ORD spectra; specific rotations at the peak of the anomalous curves at 223 nm of the d- and l-isomers were ?989 ° and +1120 °, respectively. Considering their optical purities, the actual maximum rotations of these enantiomers were calculated as ?1520 ° and +1509 °, respectively. The conditions for the reaction of l-acetylmandelyl chloride and 2-hydroxy fatty acid esters were modified to be suitable for the optical assay of 2-hydroxy fatty acid methyl esters. The method was also shown to be applicable to the assay of 3-hydroxy isomers and possibly to other positional isomers. The absolute configuration of 2-hydroxy fatty acid methyl esters obtained from calf brain cerebrosides and from yeast cerebrins were determined to be d(?), confirming previous assignments. Methyl cerebronate obtained from its less soluble strychnine salt was found to be the l(+)-enantiomer, contrary to a previous assignment. Methyl d-cerebronate was hydrolyzed with tetrahydrofuran-HCl to obtain D-cerebronic acid without racemization.     

Isolation, characterization, and esterase and CO2 hydration activities of ubiquitin from bovine erythrocytes

Ubiquitin was isolated from bovine erythrocytes by a relatively simple procedure involving extraction with chloroform and ethanol, chromatography on DEAE-cellulose, and gel filtration. Amino acid and partial sequence analyses showed it to be identical to previously isolated material. Ubiquitin released p-nitrophenolate from p-nitrophenyl acetate, but did not cleave other esterase substrates that were tested. It had a turnover number of 116 mmol for p-nitrophenyl acetate at pH 7.7 and 30 degrees C, and this activity was relatively stable to heat treatment. Electrophoretic studies indicated that the ubiquitin was sequentially acetylated by p-nitrophenyl acetate, as judged by the appearance of more anodically migrating components. The reactions of ubiquitin with p-nitrophenyl acetate at pH 7.0 were biphasic and consisted of (a) an initial phase, during which the release of p-nitrophenol resulted from monoacetylation of the ubiquitin and from ubiquitin-catalyzed hydrolysis of the ester; and (b) a second phase, during which the release of p-nitrophenol resulted only from the breakdown and reformation of the acetyl-enzyme complex. Ubiquitin also showed CO2 hydration activity and could be localized following gel electrophoresis by the CO2-bromthymol blue staining method. The strong inhibitor of carbonic anhydrase, acetazolamide, also inhibited the CO2 hydration activity and p-nitrophenyl acetate activity of ubiquitin. An antibody against this protein did not precipitate bovine carbonic anhydrase II. The esterase activity of ubiquitin was much higher than those previously reported for the carbonic anhydrases.