The acetohydroxy acid synthase III isoenzyme of Escherichia coli K-12: regulation of synthesis by leucine.

Acetohydroxy acid synthase III (AHAS III) is one of the three isoenzymes which catalyze the condensation reaction for the biosynthesis of the branched chain amino acids in $\text{Escherichia coli}$ K-12. The synthesis of this enzyme is repressed by leucine. As a consequence of this regulatory feature, strain PS1035, in which AHAS III is the only AHAS isoenzyme expressed, does not grow in minimal medium containing leucine. The other two branched chain amino acids, isoleucine and valine, do not have regulatory effects on AHAS III synthesis.     

Leucine biosynthesis: effect of branched-chain amino acids and threonine on a-isopropylmalate synthase activity from aerobic and anaerobic microorganisms

$\text{a-}\text{Isopropylmalate}$ synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and $\text{a-}\text{ketoisovalerate}$ was $\text{a-}\text{isopropylmalate}$ synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Biosynthesis of polymyxin E III. Total synthesis of polymyxin E by a cell-free enzyme system

Polymyxin E, an antimicrobial branched cyclic decapeptide, was synthesized by an enzyme fraction partially purified from crude extracts of the producing organism, $\text{Aerobacillus}\text{polyaerogenes}$. For the synthesis, three constituent amino acids (L-2,4-diaminobutyric acid, L-leucine, and L-threonine), ATP, Mg²⁺ and an acylating system consisting of octanoyl CoA and an ammonium sulfate fraction of cell extracts are required.     

Mn++-specific reactivation of EDTA inactivated alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16.

The α-isopropylmalate synthase (EC 4.1.3.12) from $\text{Alcaligenes}\text{eutrophus}\text{H 16}$ was inactivated by EDTA in a time-dependent reaction. Only the addition of Mn⁺⁺ plus dithiothreitol could restore the activity. The substrate, α-ketoisovalerate, prevented the inactivation; the feedback inhibitor, leucine, and it's antagonist, valine, increased the rate of inactivation. Except for α,α′-bipyridyl, chelating reagents, other than EDTA had no effect on the enzyme stability. It is suggested that the α-isopropylmalate synthase is a metallo enzyme - the evidence points to Mn⁺⁺ as the metal ion - and that this enzyme uses a mechanism of catalysis which differs from that of the analogous malate synthase (EC 4.1.3.2) and citrate synthase (EC 4.1.3.4).     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Evidence for existence and a tentative identification of coenzyme in yeast thiamine pyrophosphokinase.

Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from $\text{Saccharomyces cerevisiae}$ was found to require the presence of a non-protein, non-metal compound for its activity. $\text{myo}$-Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with $\text{myo}$-inositol 1-pyrophosphate. A sample of synthetic $\text{myo}$-inositol 1-pyrophosphate was made and its coenzyme activity was observed.     

Induction of fatty aldehyde dehydrogenase activity during the development of bioluminescence in Beneckea harveyi.

Bioluminescence rises very rapidly in the later stages of growth of $\text{Beneckea harveyi}$ due to the induction of luciferase activity. This enzyme catalyzes the $\text{in vitro}$ oxidation of FMNH₂ and a long chain aliphatic aldehyde resulting in the emission of light. The present experiments report the discovery of an aldehyde dehydrogenase in $\text{Beneckea harveyi}$ which is remarkably similar to luciferase in its specificity for long chain aliphatic aldehydes. Furthermore, the activity of this enzyme is shown to be induced at the same time as luciferase thus providing strong evidence for a functional implication of aldehyde dehydrogenase in the bioluminescent system of $\text{Beneckea harveyi}$.     

Mandelic acid-4-hydroxylase, a new inducible enzyme from Pseudomonas convexa

A soluble fraction of $\text{Pseudomonas convexa}$ catalyzed the hydroxylation of mandelic acid to $\text{p}$-hydroxymandelic acid. The enzyme had a pH optimum of 5.4 and showed an absolute requirement for Fe²⁺, tetrahydropteridine, NADPH. $\text{p}$-Hydroxymandelate, the product of the enzyme reaction was identified by paper chromatography, thin layer chromatography, UV and IR-spectra.     

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, $\text{sugary}$ and $\text{amylose-extender}$, showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from $\text{sugary}$ kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by $\text{sugary}$ branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of $\text{amylose-extender}$ kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of $\text{amylose-extender}$ maize, but this activity was regenerated by the addition of any branching enzyme.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Adentylate cyclase activity in myocardium of spontaneously hypertensive rat: effect of endogenous factors and solubilization

The isoproterenol- and glucagon-stimulated adenylate cyclase activities in the myocardial membranes of hypertensive rat were consistantly lower as compared with normal controls. Addition of cytosolic fraction (100,000 x$\text{g}$ supernatant) to the particulate preparation had an additive effect for glucagon and Gpp(NH)p stimulated enzyme activity and a synergistic effect for isoproterenol stimulation. Cytosolic fraction of normal control animals did not bring the adenylate cyclase activity in SHR equivalent to the control values. The basal and F^?-stimulated enzyme activity of solubilized adenylate cyclase was reduced by about 30% in SHR as compared with WKY, which could be due to a decrease in the actual amount of adenylate cyclase in the myocardium of SHR.     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene

A procedure is described for the isolation of cytochrome P-450 fraction from hamster liver microsomes. It involves removal of NADPH-cytochrome c reductase activity by treatment with bacterial protease before solubilization with Triton X-100 and precipitation with ammonium sulfate. Reconstitution studies indicate that 2-acetylaminofluorene $\text{N}$-and ring-hydroxylation require both cytochrome P-450 fraction and the reductase fraction. $\text{N}$-hydroxylation activity of cytochrome P-450 fraction from 3-methylcholanthrene pretreated hamsters is different and severalfold greater than that of cytochrome P-450 fraction from controls. These results demonstrate for the first time an activation of a chemical carcinogen by a reconstituted cytochrome P-450 enzyme system.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

Phosphorylation of tyrosine aminotransferase in vitro by cyclic nucleotide-dependent protein kinases

An intraperitoneal injection of either leucine (1.57 mg/g body wt) or valine (2 mg/g body wt) into newborn mice led to a rapid accumulation of inactive monoribosomes in their brains. $\text{In}\text{vitro}$ measurements of protein synthesis by the remaining active ribosomes in leucine-treated mice revealed that polypeptide chain elongation was also inhibited. When a mixture of the seven amino acids from the leucine transport system was injected (0.15 mg each amino acid/g body wt) following the valine or leucine treatment, brain monoribosomes did not accumulate and elongation rates in the leucine-treated mice were only slightly altered.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

A mutation affecting the valine sensitivity of the acetohydroxyacid synthase III isoenzyme in E. coli K-12

The $\text{ilv-751}$ mutation (obtained by $\text{mu}$ phage mediated mutagenesis) affects the sensitivity to valine inhibition of the acetohydroxy acid synthase III isoenzyme of $\text{E. coli}$ K-12, as shown by constructing multiple mutants containing the $\text{ilv-751}$ mutation and only one of the genes for the expression of the three acetohydroxy acid synthase isoenzymes, at once. The mutation is dominant. This suggests that the phenotype of $\text{ilv-751}$ mutation is not caused by inactivation of a gene concerned with the expression of the AHASIII enzyme, consequent to prophage insertion into that locus.     

Bacteriophage-induced lytic enzyme which hydrolyzes L-alanine-D-glutamic acid peptide bond in peptidoglycan

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.