Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells.

Four toxins have been isolated by Sephadex G-15 and high pressure ion exchange chromatography from the soft shell clams, $\text{Mya arenaria}$, which were collected during 1972 and 1974 red tide outbreaks on the New England coast. One of the toxins was saxitoxin and the rest seem to be new toxins. The same toxins were isolated from the extract of cultured $\text{Gonyaulax tamarensis}$.     

Novel nucleotides from E.coli isolated and partially characterized

Two new nucleotides have been found in the formic acid extracts of $\text{Escherichia}\text{coli}$, $\text{Clostridium}\text{botulinum}$, $\text{Bacillus}\text{subtilis}$ and $\text{Rhodospirillum}\text{rubrum}$ isolated during log phase growth. In $\text{E.}\text{coli}$ the compounds are present at all times during cell growth but increase in amount during interruption of aeration and transition to stationary phase. They migrate close to ppGpp during one dimensional chromatography on PEI cellulose but are clearly separated from ppGpp by paper chromatography. The compounds are unstable on PEI cellulose and purification was effected by chromatography on A25 Sephadex ion exchange columns. Preliminary characterization indicates that the predominant compound is a dinucleoside polyphosphate and that both compounds contain a modified adenosine nucleoside.     

Quantitative analysis of pyridine nucleotides in red blood cells: a single-step extraction procedure.

Quantitative analysis of red cell pyridine nucleotides has been unreliable in the past because of technical problems in extracting them in the presence of hemoglobin. A simple alcoholic extraction procedure for analysis of pyridine nucleotides in red blood cells is described in this paper. Pyridine nucleotides extracted in the presence of hemoglobin in solution show recoveries of NADH, NAD, and NADP averaging over 70%, while recoveries of NADPH were about 60%. In order to show that these techniques could detect actual intracellular differences in nucleotides inside red cells, two experiments were performed in which the ratios of the nucleotides would be predictably altered. Intact cells incubated in the presence of methylene blue show a decrease in the $\text{NADPH}\text{NADP}$ ratio, and intact cells incubated in the presence of hydrazine and lactate show an increase in the $\text{NADH}\text{NAD}$ ratio. The changes in pyridine nucleotide ratios in these experiments are in the expected direction and were easily detected. Levels of pyridine nucleotides in red blood cells of normal human adults are also presented.     

A novel method for measurement of the specific radioactivity of gamma-32P ATP.

The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-³²P] ATP to [1-³H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ${}^{32}\text{P}{}^3\text{H}$ ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-³²P] ATP in biological samples.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

Low molecular weight mitogenic factor produced by BRL-3A cultured rat liver cells

A new mitogenic factor has been isolated from medium conditioned by BRL-3A rat liver cells. The factor has been partially purified by a two step procedure involving ion exchange chromatography on Dowex 50 followed by gel filtration chromatography on Sephadex G-75 in 1 M acetic acid. The factor is eluted from the Sephadex G-75 column in the low molecular weight region, behin three peaks of multiplication stimulating activity. The factor is inactivated by treatment with trypsin and dithiothreitol, suggesting that it is a peptide that contains a disulfide linkage. Unlike multiplication stimulating activity, the new factor only weakly stimulates DNA synthesis in quiescent chick fibroblasts, whereas it strongly stimulates DNA synthesis in quiescent NIL8 hamster cells, $\text{BALB}\text{c}$ 3T3 cells, and IMR-90 human fibroblasts.     

Inosine from liver as a possible energy source for pig red blood cells

Adult pig red blood cells are unable to metabolize glucose due to a membrane permeability barrier to glucose developed shortly after birth. $\text{In}$$\text{vitro}$, pig red cells incubated in their own plasma are unable to maintain normal ATP levels, thus the question has been raised as to the nature of the metabolic energy source. We have suggested that organs, such as the liver, might supply low levels of substrate to the red cells as they transit through the organ. In this paper, evidence is presented to show that perfused pig livers supply a metabolic substrate used by pig red cells. This substrate has been tentatively identified as inosine.     

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.     

A role of cathepsin B1 in polymorphonuclear leukocytes chemotaxis.

Cathepsin B_I¹ was purified from rat liver lysosomal fraction by ammonium sulfate fractionation, followed by chromatography on Sephadex G-200 and DEAE-Sephadex. Formation of chemotactic factor for guinea pig polymorphonuclear (PMN) leukocytes was demonstrated $\text{in vitro}$ when guinea pig serum was incubated with cathepsin B_I. This factor formation was dependent on SH-reagent dithiothreitol (DTT) and was maximal at pH 6.0. ZnSO₄, an inhibitor of cathepsin B_I, inhibited the chemotactic factor formation likewise.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Modulation of glucose transport in human red blood cells by ATP

Depletion of energy stores of human red cells decreases the maximum transport capacity, $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$, for glucose transport to a value one-third or less of that found in red cells from freshly drawn blood. There is no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. Hemolysis and resealing of red cells with ATP or ADP reverses the decrease in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$. The maximum effect occurs at concentrations of ATP in the normal range for red cells, however, there is little effect from ADP concentrations in its normal range in freshly drawn red cells. Hemolysis and resealing with ATP gives an increase in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$ and an increase in differential labeling by photolytic labeling with tritiated cytochalasin B. Most of the activation is lost after a second hemolysis-reseal without ATP but about 25% of the activation remains.     

Identification and purification of a soluble adenosine triphosphatase from Tetrahymena pyriformis .

An unusual ATPase isolated from the postribosomal supernatant fraction of $\text{Tetrahymena pyriformis}$ has been purified to homogeneity. The purification procedure consisted of protamine sulfate and heat treatment; column chromatography successively on phosphocellulose, DEAE-cellulose and Sephadex G-150; and isoelectric focusing. The pure enzyme has a molecular weight of 89,000 and requires either Ca²⁺ or Ba²⁺ for maximum activation. Nucleoside triphosphates are hydrolized at decreasing rates in the order: ATP > GTP > ITP > CTP > UTP. The K_m for ATP is 2.5 mM. Because of its properties the enzyme is tentatively classified as a soluble Ca²⁺-activated ATPase.     

Measurement of phosphate esters in extract of fetal rat heart by automated high pressure liquid chromatography

Creatine phosphate, nucleotides and glycolytic phosphate esters were estimated in extract of beating, in situ freeze clamped, $\text{131}\text{2}$ to $\text{191}\text{2}$ day fetal rat hearts by automated phosphate ester chromatography. Creatine phosphate increased more than 4-fold to almost 9 n moles per mg. protein at $\text{191}\text{2}$ days, while ATP remained relatively constant at about 19 to 21 n moles per mg. protein. Most other nucleotides decreased as gestation advanced. ATP rather than creatine phosphate appears to be the major energy source of fetal rat heart. Except for glucose-6-phosphate, which increased, the glycolytic phosphate esters decreased only very slightly with advancing gestational age, suggesting a relatively stable basal glycolytic activity. Methodology includes correction for phosphate esters of whole blood trapped in extracts of in situ freeze clamped tissues.     

Modulation of the root effect in goldfish by ATP and GTP

Both ATP and GTP are present in considerable amount in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2–6 times more effective than ATP.The two triphosphates account for $\text{3}\text{4}$ of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH.     

Femtomole level of analysis of biogenic amines and amino acids using functional group mass spectrometry

A general method for the determination of compounds possessing either the primary amine structure, R-CH₂-NH₂ (I), or the α-amino acid structure, RCHNH₂COOH (II), has been devised using gas chromatography and mass spectrometry. Trimethylsilyl derivatives of the biogenic amines (phenylethylamines, indoleethylamines, or Ω-amino acids) produce an intense ion at $\text{m}\text{e}$ 174 upon fragmentation; TMS derivatives of α-amino acids produce an ion at $\text{m}\text{e}$ 218. For maximum sensitivity, chromatograms were obtained with the mass spectrometer tuned to detect a single ion fragment characteristic of a group of structurally related compounds (i.e., functional group GC-MS). At $\text{m}\text{e}$ 174 up to 14 compounds of Type I, including glycine, γ-aminobutyric acid, dopamine, and 5-hydroxytryptamine, could be determined in a single analysis. Detection limits range from 10–100 femtomoles (10^?15 moles). At $\text{m}\text{e}$ 218, eight compounds of Type II, including isoleucine, phenylalanine, tyrosine, and DOPA could be determined. This technique has been applied to the assay of these compounds in extracts containing 0.1 mg mouse brain or abdominal ganglia of the marine molluse, Aplysia californica.     

The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent

Trimethyloxonium ion inactivates acetylcholinesterase from the electric eel and acetylcholinesterase on the surface of human red blood cells. Tetramethylammonium ion, which is a competitive inhibitor of acetylcholinesterase, protects against this inactivation. Trimethyloxonium ion does $\text{not}$ inactivate the system that transports choline into the red blood cell. We conclude that trimethyloxonium ion is an affinity-labeling reagent for acetylcholinesterase and that red blood cell acetylcholinesterase is probably not a component of the choline transport system.     

Differences in the physical properties of collagenases isolated from rheumatoid synovium and human skin

During purification of human collagenase from normal skin and rheumatoid synovium differences have been observed with regard to the size and charge properties of the two enzymes. Gel filtration experiments in Sephadex G-100 superfine have allowed molecular weights of approximately 63,000 and 32,000 daltons to be calculated for the skin and rheumatoid synovial enzyme respectively. Ion exchange chromatography using Sephadex QAE, A-50 has shown the rheumatoid synovial enzyme to possess charge properties more basic than that of the skin enzyme. Elution of collagenase activity from $\text{7}\text{1}\text{2}\text{\%}$ polyacrylamide gels has produced no evidence for a ‘fast-moving’ component of either enzyme.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca2+-loading

Human red cells (RBC) respond to moderate Ca²⁺-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca²⁺-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase ($\text{GAPDH}\text{PGK}\text{)-steps}$. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca²⁺-extrusion rate and not to the loaded Ca²⁺. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca²⁺-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the $\text{GAPDH}\text{PGK}$ equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. $\text{NAD}\text{NADH}$ ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.     

A dialyzable protein synthesis inhibitor released by mammalian cells in vitro

Macrophages, lymphocytes, and HeLa cells when incubated $\text{in vitro}$ at 37° in Krebs-Ringer phosphate buffer release a dialyzable, heat-stable, ninhydrin-reacting factor which inhibits protein synthesis by intact cells and isolated ribosomes. Release of the inhibitor appears to be dependent on metabolism. Partial purification of the inhibitor by Sephadex G-10 chromatography suggests it has a molecular weight of 400–600.