The presence and photoregulation of protochlorophyllide reductase in green tissues

Summary The enzyme protochlorophyllide (pchlide) reductase has been identified amongst the peptides, resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), of chloroplast membranes from oat and barley plants. In support of this identification the enzymic activity associated with the enzyme has also been measured in the same preparations. A higher level of enzyme was found in plants which had been darkened prior to extraction. Based on this data, mechanisms for the light regulated diurnal variation of the reductase are discussed.     

Ultrastructure of intestinal and gall-bladder epithelium in the teleost Gasterosteus aculeatus L., as related to their osmoregulatory function

Summary Intestinal and gall-bladder epithelial cells in sticklebacks have been examined in ultrathin sections and freeze-etch replicas. Enterocytes throughout the intestine appear to have a well-developed basal labyrinth similar to that of renal tubular cells, consisting of baso-lateral infoldings closely associated with numerous mitochondria. The lumen inside these intracellular membranes is continuous with the intercellular space via pores. Such a membrane system is also present in the epithelial cells lining the gall bladder, distinguishing them from gall-bladder cells of higher vertebrates. Morphometric analysis indicates that the basal labyrinth of enterocytes in the posterior part of the intestine increases markedly in both sexually mature males and androgen-treated females. This does not occur in the anterior part or gall bladder. In sticklebacks, androgens cause reduced urine excretion and enhanced fluid release via the anus. We conclude that the cells lining the intestine and gall bladder possess an extensive basal labyrinth that may function as a backward channel system, enabling fluid to be produced in the intestine of fish. The androgen-induced increase in the extent of the basal labyrinth in the posterior part of the intestine may be related to the enhanced rate of intestinal fluid excretion observed in sexually mature male sticklebacks.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

The inhibition of acetate oxidation by ethanol in Acetobacter aceti

Ethanol grown Acetobacter aceti differed from acetate grown. In ethanol grown cells, acetate uptake, caused by the oxidation of acetate, was completely inhibited by ethanol, in acetate grown cells only to 20%. This was correlated with a 65-fold higher specific activity of the membrane bound NAD(P)-independent alcohol dehydrogenase in ethanol grown than in acetate grown cells. In comparison with ethanol grown cells, acetate grown cells showed a 3-fold higher acetate respiration rate and 3-fold higher specific activities of some tricarboxylic acid cycle enzymes tested. Both adaptations were due to induction by the homologous and not to repression by the heterologous growth substrate. A. aceti showed a membrane bound NAD(P)-independent malate dehydrogenase and no activity of a soluble NAD(P)-dependent one, as was known before from A. xylinum. A hypothesis was proposed explaining the observed inhibition of malate dehydrogenase and of functioning of the tricarboxylic acid cycle in the presence of ethanol or butanol or glucose by a competition of two electron currents for a common link in the convergent electron transport chains. The electrons coming from the quinoproteins, alcohol dehydrogenase and glucose dehydrogenase on the one side and those coming from the flavoproteins, malate dehydrogenase and succinate dehydrogenase via ubiquinonecytochrome c reductase on the other side are meeting at cytochrome c. Here the quinoproteins may be favoured by higher affinity and so inhibit the flavoproteins. Inhibition could be alleviated in the cell free system by increasing the oxygen supply.Dedicated to Professor Carl Martius on the occasion of his 80th birthday, March 1st 1986     

The role of electrogenic xylem pumps in K+ absorption from the zylem of Vigna unguiculata: the effects of auxin and fusicoccin

Abstract We tested the hypothesis that electrogenic ion pumps, working at the parenchyma symplast/xylem interface of pea hypocotyls, provide the driving force for K⁺ uptake from the xylem. Solutions of known composition were perfused through a hypocotyl segment. The K⁺ activity of the solution flowing out of the xylem (K⁺_out) increased (i.e. K⁺ uptake decreased) when aerobic respiration was inhibited by lack of O₂, and this was preceded by a decrease in V_px (electrical potential difference between parenchyma symplast and xylem). Perfusion with auxin (1AA) and fusicoccin (FC) stimulated the electrogenic activity of the ‘xylem pumps’ (111 and 205% respectively) and stimulated uptake of K ⁺ from the xylem (with 71% and 29% respectively). The close correlation between xylem pump activity and K⁺ uptake corroborated the aforementioned hypothesis. Interestingly, inhibition of pump activity by anoxia was incomplete in the presence of FC. It is thought that FC increases the affinity of the ATP-requiring xylem pump for ATP, thus ensuring that ATP production during fermentation is sufficient to fuel the pump in the absence of O₂.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina

Abstract: Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca²⁺-binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca²⁺ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5′-(β,γ-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn²⁺-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.     

Hormonal Regulation of Adipose S-100 Protein Release

The release of S-100 protein from epididymal fat pads was enhanced by epinephrine in vitro, and about 50% of S-100 protein in the tissue was released into the medium after 2-h incubation at 37 degrees C with 10 microM epinephrine. Similar results were obtained with the incubation of isolated adipocytes. The S-100 protein release was also enhanced by isoproterenol, norepinephrine, ACTH, and dibutyryl cyclic AMP, which all increase the lipolysis by increasing cyclic AMP levels in the tissue. Propranolol, a beta-adrenergic blocker, could block the increase of S-100 protein release by catecholamines, indicating that the release was mediated by the beta-adrenergic effect of catecholamines. However propranolol had no suppressive effect on the enhancement of S-100 protein release by ACTH or dibutyryl cyclic AMP. Insulin had an inhibitory effect on the epinephrine-enhanced S-100 protein release. Epinephrine or ACTH could not stimulate the S-100 protein release in the absence of Ca2+, whereas the epinephrine-enhanced glycerol release was not affected under the same conditions. The increase in S-100 protein release was induced by only a pretreatment of the tissue with epinephrine. However, the lipolysis in the tissue was not enhanced by the pretreatment alone. These results indicate that the release of S-100 protein from adipocytes is regulated by the hormones that have been known to control the lipolysis with a manner slightly different from that of lipolysis.     

Murine “housekeeping” enzyme (genetic locus:Idh-1) is regulated in an allele-specific manner

The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1^a allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1^a allele. While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.