Interdependence of glucose and arginine catabolism in Streptococcus faecalis R. ATCC 8043

The growth of $\text{Streptococcus faecalis}$ R.ATCC 8043 was found to be dependent on the simultaneous presence of both glucose and arginine, the molar ratio of utilization of these nutrients being 1:1. The growth coefficient (Y-glucose or Y-arginine) was near about 30 during exponential phase suggesting the generation of 3 moles of ATP during glycolysis. Glycolytic activity in cells was directly proportional to the intracellular pool of either arginine or citrulline and in aged cells the loet glycolytic activity could be restored by the addition of arginine to thecell suspension. Cell free extract of $\text{S}$. $\text{faecalis}$ was found to transfer phosphate group from carbamyl phosphate (a catabolic product of arginine) to glucose.     

Porphyrin mutants of Saccharomyces cerevisiae: Correlated lesions in sterol and fatty acid biosynthesis

The $\text{ole2}$, $\text{3}$ and $\text{4}$ mutants of yeast require an unsaturated fatty acid and methionine for growth and do not synthesise ergosterol. They have very similar sterol compositions and all accumulate lanosterol. The mutants lack cytochrome pigments and have negligible respiratory activity. Porphyrin intermediates alleviate the lipid requirement of $\text{ole2}$ and $\text{ole3}$ and restore respiratory competence. It is concluded that the primary defects in these mutants are lesions in porphyrin biosynthesis.     

Escherichiacoli mutants deficient in exoribonucleases

Strain S296, isolated by screening 2000 colonies after nitrosoguanidine mutagenesis, yields extracts with less than 1% of wild-type RNase activity against (³H) poly(U). Unlike other $\text{E.}\text{coli}$ strains, S296 grows with a doubling time of about 2 hr., both in nutrient broth and in minimal medium, and at 30°, 37° and 42°. The strain retains 10 to 20% of wild-type exonuclease activity against (³H) rRNA or T4 phage-specific mRNA; but two further mutants, made by screening mutagenized colonies of strain S296, are reduced to 3% of wild-type activity against those substrates as well.     

The p-cymene pathway in PseudomonasputidaPL: Isolation of a dihydrodiol accumulated by a mutant

A mutant strain (PL pT $\text{11}\text{43}$) of $\text{Pseudomonas}\text{putida}$ PL, has been isolated for its inability to growth with $\text{p}$-cymene as carbon source. The mutant oxidizes $\text{p}\text{-cymene}$ (and $\text{p}$-cumate) to a compound (λmax 293 nm) which is readily converted to 3-hydroxy-$\text{p}$-cumate by acid. 4-Trifluoromethylbenzoate is oxidized by the mutant to an acid-stable intermediate (λmax 277nm) that has been crystallized. The spectral properties (u.v., i.r., NMR and mass) of this metabolite are consistent with those expected for a 2,3-dihydro-2,3-dihydroxy derivative of 4-trifluoromethylbenzoate. Further support of this structure was provided by elemental analysis and the properties of two derivatives of the metabolite, 4-trifluoromethyl-3-hydroxybenzoate and an acetonide formed with 2,2-dimethoxypropane. The stability of a product obtained by treatment of the dihydrodiol metabolite with triacetylosmate indicates that it is the $\text{cis}\text{-isomer}$.     

Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli

Spermidine acetylase activity was detected in extracts prepared from $\text{Escherichia coli}$ and there was a marked increase in activity over the early period of growth. This increase reached a maximum 3 h after inoculation and was followed by an increase in ornithine decarboxylase activity. The acetylase was also able to use spermine as a substrate, but not putrescine. With spermidine and acetyl-CoA as substrate, the product formed was exclusively N¹-acetyl-spermidine. This is the first evidence for the occurrence in bacteria of spermidine-N¹-acetyltransferase, an enzyme which has previously been described in mammalian cells. These results suggest that acetylation of spermidine may be involved in the growth of $\text{Escherichia coli}$ and in the regulation of its polyamine content.     

Biological potency of synthetic somatostatin in the oxidized and reduced form on the inhibition of secretion of growth hormone

The reduced and oxidized forms of synthetic somatostatin are equipotent to inhibit the secretion of immunoreactive growth hormone when tested $\text{in vitro}$ and $\text{in vivo}$ by intravenous administration. The reduced peptide has approximately half the potency and twice the duration of biological activity of the oxidized form when both are administered subcutaneously.     

Ephedrine reduces weight of viable yellow obese mice (Avy/a)

Ephedrine administered subcutaneously or in the diet reduces the weight of viable yellow obese mice ($\text{A}{}^{\mathrm{vy}}$/$\text{a}$) and normal mice. The compound has some appetite-suppression activity; however, reduced food consumption could not account for all of the weight loss. Ephedrine did not affect lipogenesis or lipolysis acutely. The major portion of the weight loss was due to the loss of triacylglycerol. The rectal temperature of long-term treated $\text{A}{}^{\mathrm{vy}}$/$\text{a}$ mice was elevated suggesting that ephedrine may alter energy metabolism.     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

A soybean lectin having 4-O-Methyl-D-Glucuronic acid specificity

Evidence is presented for the presence of a new lectin activity in soybean seeds [$\text{Glycine}\text{max}$ (L.) Merrill] that has specificity towards the 4-O-methyl-$\text{D}$-glucurono-$\text{L}$-rhamnan exopolysaccharide produced by certain strains of $\text{Rhizobium}\text{japonicum}$. Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the “lectinless” varieties. Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-$\text{O}$-methyl-$\text{D}$-glucuronic acid, $\text{D}$-glucuronic acid and their methyl glycosides.     

Spectral properties of a cyanobacterial cytochrome c oxidase: Evidence for cytochrome a.a3

Membranes isolated from $\text{Nostoc}$ sp. strain Mac oxidised NAD(P)H and horse heart ferrocytochrome c in dark reactions inhibited by KCN, NaN₃, CO, and by anaerobiosis. Reduced $\text{minus}$ oxidised difference spectra revealed peaks at 603 and 445 nm which shifted to 590 and 430 nm, respectively, in reduced $\text{plus}\text{CO}\text{minus}$ reduced spectra. In presence of suitable electron mediators the pigment could be reduced also with NAD(P)H or ascorbate; KCN prevented this reduction. Photoaction spectra of CO-inhibited membranes showed peaks at 590 and 430 nm. From the results it is concluded that cytochrome a.a₃ is a functional respiratory oxidase in $\text{Nostoc}$ sp. strain Mac.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Light-dependent development of photosynthetic competence in Scenedesmus mutant no. 8

The heterotrophically grown, $\text{P-700-}\text{free}$ mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or $\text{P-700}$ turn-over could not be detected.When grown mixotrophically, the mutant showed increasing $\text{P-700}$ activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good $\text{P-700}$ turn-over and reasonable rates of NADP reduction.The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of $\text{P-700}$ in linear electron transport.     

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

Purification of endo-(1→3)-β-D-glucanases lysing yeast cell walls from Rhizoctonia solani

Three forms of $\text{endo}\text{-(1→3)-β-}\text{g}\text{-glucanases}$ lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of $\text{endo}\text{-(1→3)-β-}\text{D}\text{-glucanases}$ lysing yeast cell walls.     

(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.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.     

Kinetic studies on the inhibition of (Na+ + K+)-ATPase by diketocoriolin B

Diketocoriolin B, a sesquiterpene antitumor antibiotic, inhibits particulate $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) of Yoshida sarcoma cells competitively, with respect to ATP, and uncompetitively with respect to Na⁺ and K⁺. The inhibition is reduced by the addition of phosphatidylserine.Rat brain $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$, which is solubilized by deoxycholate and requires phosphatidylserine for its activity, is also inhibited by diketocoriolin B competitively with respect to ATP and the inhibition was reversed by increasing the concentration of phosphatidylserine.However, several differences are found between the solubilized and particulate systems: (a) 2 moles of diketocoriolin B interact with the former, while only one mole interacts with the latter, (b) K⁺-dependent phosphatase activity of the former requires phospholipid and is sensitive to diketocoriolin B while the reverse is true with the latter.Based on these kinetic studies, it is supported that $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ has two binding sites for phospholipid, one being essential for K⁺-dependent phosphatase activity and when these two sites are filled with the appropriate phospholipids, ATP can bind to the enzyme.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.