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.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

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.     

The regulation of ferredoxin-dependent nitrogenase activity in Rhodospirillum rubrum and Rhodopseudomonas capsulata

Abstract The regulatory properties of Rhodospirillum rubrum nitrogenase reduced by either the endogenous electron donor (ferredoxin) or an artificial donor (dithionite) were examined. The nitrogenase obtained from glutamate-grown cells required activating enzyme for maximum activity with either reductant. The activating enzyme requirement of ferredoxin-dependent nitrogenase activity implies a physiological significance of the activating enzyme in R. rubrum. Rhodopseudomonas capsulata nitrogenase also required activating enzyme when dithionite was the reductant, but there appeared to be no activating enzyme requirement with ferredoxin as the reductant. Because the catalytic activity of the enzyme was very low under these conditions, the physiological significance of activating enzyme in this organism remains in question.     

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