Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.     

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.     

Catecholaminergic regulation of the hypothalamic-pituitary-adrenocortical activity.

The significance and site of adrenergic receptors involved in the control of the hypothalamic-pituitary-adrenal axis (HPA) activity was assessed indirectly by estimation of serum corticosterone levels 1 h after drug administration to conscious rats. Adrenergic drugs were given intracerebroventricularly (icv) and intraperitoneally (ip), the antagonists 15 min prior to the agonists. Noradrenaline, adrenalin and isoproterenol given by either route increased dose-dependently the serum corticosterone levels. The corticosterone response to icv noradrenaline was almost abolished by icv pretreatment with propranolol, a beta-adrenergic antagonist, and yohimbine, and alpha 2-receptor blocker, and was also considerably reduced by prazosin, an alpha 1-adrenergic antagonist. When given ip, these antagonists did not significantly influence the noradrenaline induced corticosterone response, which suggests a suprapituitary site of action of noradrenaline in stimulation of the HPA. The corticosterone response to icv adrenalin was suppressed by prazosin given by either route. The corticosterone response to ip adrenalin was almost abolished by pretreatment with yohimbine, and also significantly diminished by propranolol given by the same route. The increase in corticosterone secretion, induced by isoproterenol given by either route, was abolished by ip injection of propranolol. These results indicate that noradrenaline stimulates the HPA via alpha- and beta-adrenergic receptors, mainly at the suprapituitary level. Adrenalin increases that activity both via central and pituitary alpha- and beta-adrenoceptors. Isoproterenol activates the HPA by stimulation of pituitary beta-receptors.     

Histamine-releasing activity. IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA approximately 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.     

Estrogen-dependent trypsin-like activity in the rat uterus. Localization of activity in the 12,000g pellet and nucleus.

Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treatment, indicating organ specificity. The enzyme (or enzymes) from the 12,000g pellet was solubilized and some characteristics were determined. The apparent K_m is about 1.0 × 10^?6m, the temperature optimum is about 44 °C and maximum velocity is achieved in the alkaline range (pH ~ 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin.     

Measuring the potential activity of hydrocarbon-degrading bacteria.

[14C]hydrocarbons were utilized as a means of estimating the hydrocarbon-degrading potential of bacteria in estuarine and marine environments. Evaporation of the hydrocarbons must be considered in estimates of oxidation. Amount of mineralization of [14C]hexadecane can be equated with the total number of petroleum-degrading bacteria and the percentage of the total heterotrophic population, which they represent. Mineralization activity was found to be related to the activity of the bacterial populations during in situ incubation. Rates of mineralization were observed, as follows, for [14C]hexadecane greater than [14C]naphthalene greater than [14C]toluene greater than [14C]cyclohexane. Increased rates of uptake and mineralization were observed for bacteria in samples collected from an oil-polluted harbor compared with samples from a relatively unpolluted, shellfish-harvesting area, e.g., turnover times of 15 and 60 min for these areas, respectively, using [14C]hexadecane.