DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH₂-Prothrombin-COOH $\text{Xa or thrombin}$ NH₂-Intermediate 3 + Intermediate 1-COOH; NH₂-Intermediate 1-COOH $\text{Xa}$ NH₂-Intermediate 4 + Intermediate 2-COOH; NH₂-Intermediate 2-COOH $\text{Xa}$ NH₂-A chain α-thrombin -S-S-B chain α-thrombin-COOH.Previous reports from this laboratory have demonstrated that the activation of prothrombin proceeds through several single-chain intermediates prior to the appearance of thrombin activity. (1) Subsequent studies have sequence of the prothrombin molecule can be deduced from the sequences of its activation intermediates and we are continuing our studies toward this goal.     

Selective fetal malnutrition: The effect of ethanol and acetaldehyde upon in vitro uptake of alpha amino isobutyric acid by human placenta

Uptake of alpha amino isobutyric acid was measured in human placental villus tissue exposed $\text{in vitro}$ to ethyl alcohol (ethanol) (0.3 g/dl–2 g/d1) or acetaldehyde (50 μM-20 mM). Ethanol and acetaldehyde significantly inhibited uptake of amino acid at higher, pharmacologic concentrations (2 g/dl and 2–20 mM respectively). Inhibition by 10 mM acetaldehyde was partially reversible. The results suggest that the human placenta is resistant to acute ethanol-associated effects upon amino acid transport $\text{in vitro}$. However, both ethanol and its major circulating metabolite, acetaldehyde, may still alter placental function during $\text{in vivo}$ chronic exposure.     

Structural studies on epsilon-prototoxin of Clostridium perfringens type D. Localization of the site of tryptic scission necessary for activation to epsilon-toxin.

The mechanism of activation of ε-prototoxin to ε-toxin has been ascertained from partial amino acid sequences of both ε-prototoxin and ε-toxin. The activation of ε-prototoxin from $\text{Clostridium perfringens}$ type D by brief exposure to trypsin is caused by scission of a peptide bond between Lys₁₄ - Ala₁₅. A small peptide (14 amino acid residues) is split from the NH₂-terminus of the ε-prototoxin to give the active ε-toxin.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.     

Effect of phenethylbiguanide on sugar and amino acid transport in rabbit ileum

Phenethylbiguanide has been shown to be an inhibitor of sugar and amino acid uptake in both $\text{in vivo}$ and $\text{in vitro}$ conditions. This action could be due to a competition for sodium sites on the sugar and amino acid carrier molecules. The effects of phenethylbiguanide on $\text{in vitro}$ intestinal preparations indicate that this compound has a time-dependent effect, it is most effective when placed on the mucosal surface but is also effective on the serosal surface. Furthermore, competition studies indicate that it is a competitive inhibitor of sugar uptake and a non-competitive inhibitor of amino acid uptake. These results are consistent with the differences in the mechanism of coupled transport between sugars and amino acids, but, do not substantiate the idea that phenethylbiguanide competes for the sodium site on the ternary carrier.     

Transport of arginine by an in vitro system

Cell surface glycoproteins of $\text{Neurospora crassa}$ conidia have been shown to bind amino acids and to be genetically associated with the previously defined amino acid transport systems of that organism. L-arginine does not readily permeate a film of $\text{Neurospora}$ conidial lipids. Addition of glycoprotein extracts from $\text{Neurospora}$ to the lipid film enhances permeation of arginine at an initial rate 1000 times the rate of permeation through lipid alone. The initial rate of passage exceeds the rate of unhindered passage (no lipid film) through the same cross sectional area by 10 fold.     

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.     

Studies on the purification and properties of teleost prolactin

A protein fraction containing prolactin activity from the pituitary tissue of a teleost fish, $\text{Tilapia mossambica}$, has been purified by a combination of ion-exchange and exclusion chromatographic procedures. The purified $\text{Tilapia}$ prolactin was characterized by disc gel electrophoresis, amino-terminal group identification, and amino acid analysis. Its amino acid composition was found to be similar to ovine prolactin. The purified fish prolactin was found to be 40–50 times more potent than ovine prolactin in the $\text{Tilapia}$ sodium-retaining bioassay. However, it was found to be devoid of $\text{Gillichthys}$ yellow pigment-dispersing activity which was previously thought to be a property of teleost prolactin.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Structure and properties of hemocyanins. XIV. Reassociation of Helix pomatia alpha-hemocyanin

Helix pomatia α-hemocyanin dissociates within minutes into $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules, on increase of pH in the alkaline region.The rate and final state of reassociation of $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules have been studied, by turbidity measurements and ultracentrifugation, on lowering of pH or on the addition of calcium ions.Reassociation of $\text{1}\text{10}\text{-size}$ molecules proceeds in two phases, with half-times in the order of minutes and one hour, respectively. The slow phase is linked to the disappearance of transitory intermediates, presumably dimers and tetramers of $\text{1}\text{10}$-size molecules.A hysteresis is observed in the final state of pH-dependent and Ca²⁺-dependent dissociation-reassociation.Reassociation of $\text{1}\text{20}$-size molecules depends on the initial (isomeric) state of these molecules. The F(fast sedimenting)-$\text{1}\text{20}$-size molecules reassociate almost completely to whole molecules, whereas the S(slow sedimenting)-form does not reassociate at all.     

Structure of rhodotorucine A, a novel lipopeptide,inducing mating tube formation in Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ is a peptidyl factor which induces mating tube formation in $\text{Rhodosporidium}\text{toruloides}$. The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine $\text{A}$ was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine $\text{A}$. H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Stereochemical course of the biosynthesis of 1-aminocyclopropane-1-carboxylic acid. I. Role of the asymmetric sulfonium pole and the α-amino acid center

The substrate stereospecificity of 1-aminocyclopropane-1-carboxylic acid synthase, a pyridoxal phosphate-containing enzyme, from the pericarp tissue of $\text{Lycopersicon esculentum}$ (tomatoes) was studied using the various stereoisomers of $\text{S}$-adenosylmethionine (AdoMet) at both the sulfonium pole and the amino acid center. The data indicate that only the naturally occurring isomer (?)Ado-L-Met acts as substrate (Km = 20±5 μM). Both (±)Ado-D-Met and (+)Ado-L-Met were inactive as substrates. The (+)Ado-L-Met (Ki = 15±5 μM) was found to be a potent inhibitor of ACC synthase whereas (±)Ado-D-Met (Ki = 70±20 μM) was less active as an inhibitor. This active isomer has the ($\text{S}$) configuration at both the sulfur and the α-carbon of the amino acid portion of AdoMet.     

Enzymatic release of nitric oxide from L-alanosine,an antineoplastic antibiotic

L-Alanosine is an antineoplastic drug which is the 3-isonitramino analog of $\text{L}$-aspartic acid. The drug is known to be metabolized to the corresponding 2-oxo acid. Unlike the parent amino acid, the 2-oxo acid is unstable under mild conditions. When the 2-oxo acid is generated $\text{in}\text{vitro}$ by the aerobic action of $\text{L}$-amino acid oxidase on $\text{L}$-alanosine, the reaction mixture contains products capable of diazotizing sulfanilamide and of reducing ferricytochrome $\text{c}$ to ferrocytochrome $\text{c}$. It is thus likely that, as expected from model reactions, the unstable 2-oxo acid derived from $\text{L}$-alanosine decomposes into nitric oxide and other reactive free-radical species. Enzymatically promoted production of highly cytotoxic nitric oxide may pertain to the biological activity of the antibiotic. The reaction should prove extrapolable to the design of other enzyme-activated cytotoxic agents.     

Simultaneous determination of cysteine sulfinic acid and cysteic acid in rat brain by high-performance liquid chromatography

A sensitive and specific assay method for cysteine sulfinic acid (CSA) and cysteic acid (CA) using high-performance liquid chromatography has been developed. The method includes post-column derivatization of various amino acids with o-phthalaldehyde in the presence of 2-mercaptoethanol. The column packed with cation-exchange resin ($\text{ISC-07}\text{S1504}$, Shimadzu Sci entific instruments, Inc., Kyoto, Japan) was used for obtaining general separation of amino acids except CSA and CA, while the separation of CSA and CA was achieved using a strong-base anion exchange ($\text{ISA-07}\text{S2504}$, Shimadzu Scientific Instruments) column. The fluorescence peak area for CSA was linear between 20 pmol and 5 nmol, whereas that for CA was 10 pmol to 5 nmol. The regional distribution of CSA, CA, and other amino acids in the rat brain was studied using this new assay method.     

Inhibition of porphobilinogen synthase by heme and an enol-ether amino acid

The enol-ether amino acid, L-2-amino-4-methoxy-$\text{trans}$-butenoic acid (AMTB) is an inhibitor of porphobilinogen synthase (PBG synthase) when added prior to the addition of the substrate δ-aminolevulinic acid. The inhibition of PBG synthase by several stereoisomers and analogues of AMTB was investigated to determine those structural features of AMTB which may be necessary for inhibition. The D-$\text{trans}$ isomer was also an inhibitor after preincubation, whereas the L-$\text{cis}$ isomer inhibited with or without preincubation. The amino acid analogues, DL-vinylglycine, DL-2-aminobutanoic acid, the reduced form of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid, L-2-amino-4-(2-aminoethoxy)-$\text{trans}$-3-butenoic acid and its reduced congener did not inhibit PBG synthase even with preincubation. This structure activity relationship indicates that the $\text{trans}$ double bond and methoxy moiety of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid are probably required for inhibition.Heme, when preincubated with PBG synthase, was an inactivator of the enzyme. However, when both L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid and heme were simulatneously preincubated with PBG synthase, inactivation of the enzyme was greater than with either compound separately. The possibility of multiple catalytic sites was suggested by the use of multiple inhibition kinetics in the presence of heme and L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.