The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells. The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid. Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells. The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa. In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble). In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose. 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium. These results suggest that in S. cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the 'Ehrlich Pathway'.     

The purification and characterization of acetoacetyl-coenzyme A reductase from Azotobacter beijerinckii

A soluble acetoacetyl-CoA reductase (EC 1.1.1.36) was purified 54-fold from Azotobacter beijerinckii N.C.I.B. 9067 and the reaction product identified as d(-)-beta-hydroxybutyryl-CoA. The Michaelis constants for acetoacetyl-CoA, NADPH and NADH were determined and the reaction rate was found to be some fivefold greater with NADPH than with NADH. At neutral pH the equilibrium greatly favours the formation of the reduced product. Substrate specificity was in the order: acetoacetyl-CoA>acetoacetylpantetheine>acetoacetyl-(acyl-carrier protein). The enzyme possesses a functional thiol group, suffers inactivation by oxygen and is inhibited by thiol-blocking reagents. Inhibition by p-chloromercuribenzoate is reversed by excess of dithiothreitol, which also protects the enzyme from inactivation by oxygen.     

Glucose and fructose metabolism in Zymomonas anaerobia

Isotopic and enzymic evidence indicates that Zymomonas anaerobia ferments glucose via the Entner-Doudoroff pathway. The molar growth yields with glucose (5.89) and fructose (5.0) are lower than those for the related organism Zymomonas mobilis and the observed linear growth suggests that energetically uncoupled growth occurs. A survey of enzymes of carbohydrate metabolism revealed the presence of weak phosphofructokinase and fructose 1,6-diphosphate aldolase activities but phosphoketolase, transketolase and transaldolase were not detected. Fermentation balances for glucose and fructose are reported; acetaldehyde accumulated in both fermentations, to a greater extent with fructose which also yielded glycerol and dihydroxyacetone as minor products.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

STUDIES ON SARGASSUM. I. A LIGHT MICROSCOPIC EXAMINATION OF THE WOUND REGENERATION PROCESS IN MATURE STIPES OF S. FILIPENDULA

Regrowth from wounded stipe explants of Sargassum can be divided into four stages based on cytological changes. The first stage involves changes associated with the wound reactions and the formation of a wound epidermis. The second stage includes the formation of a well defined medullary pit with meristematically active cells around its periphery. Several “bud primordia” are also formed which begin to grow by cell division towards the wound surface. The third stage involves a period of internal tissue differentiation in the “bud primordia” such that mitotic activity is localized in the bud tip and the basal cells grow by cell elongation. The fourth stage marks a major change in the morphology of the regeneration branch from a tubular structure to that of a flattened blade. This change in morphology is preceded by the formation of an apical pit around which the flattened growth appears to be organized.     

Abnormal amino acid metabolism in mutants of Saccharomyces cerevisiae affected in the initiation of sporulation

Amino acid metabolism was examined during sporulation in a wild-type strain of Saccharomyces cerevisiae and in a mutant homozygous for the spd1 mutation (derepression of sporulation). In the wild-type initiation of sporulation involves a rise in intracellular glutamate. In the derepressed mutant there was no further increase of intracellular glutamate: glutamate was already present at a greater concentration than in the wild-type. These elevated glutamate levels are apparently due to reduction in the activity of 2-oxoglutarate dehydrogenase. One consequence of the elevated glutamate levels in diploids homozygous for the spd1 mutation is the production of considerable amounts of glycyl-L-proline, glutathione and saccharopine.     

Restriction endonuclease analysis to distinguish two closely related nuclear polyhedrosis viruses: Autographa californica MNPV and Trichoplusia ni MNPV.

Restriction endonuclease fragment patterns of the deoxyribonucleic acid genomes of Autographa californica nuclear polyhedrosis virus (multiply embedded type) and Trichoplusia ni nuclear polyhedrosis virus (multiply embedded type) demonstrate that the two viruses are distinct but closely related variants.     

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.

These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

     

Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway

The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway.