A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gels

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H₂O₂ equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe²⁺ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO₃ or SnCl₂ followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.     

Deoxynucleoside composition of DNAs and modified nucleoside composition of tRNAs determined at nanomole sensitivity by reversed-phase liquid chromatography

Selected purified tRNA and DNA samples were digested by standard enzymatic methods, and the nucleosides were separated by high-pressure liquid chromatography on reversedphase columns. Nanomole sensitivity was obtained by use of an integrator. The nucleosides were detected at wavelengths near their uv-absorption maxima, including 220 nm for dihydrouridine, and 330 nm for 4-thiouridine. Recovery values for the individual nucleosides were in the range of 94–100%. The nucleoside composition of the DNA and tRNA digests were in accurate and precise agreement with published values.     

Configurations of a polymeric antigen adsorbed to a B-cell membrane

For an unsynchronized cell population with several phases of DNA synthesis, the population distribution of tracer (thymidine) incorporation is derived. Compounding this with the Poisson probability for ³H disintegration, the distribution of autoradiographic grains over nuclei is obtained. The changes in distribution after various numbers of cell divisions are obtained on the assumption that the tracer is (approximately) equally partitioned between daughter cells during division. The parameters are expressed in terms of the number of phases of synthesis, specific activity of tracer, rate of incorporation, and autoradiographic exposure time. Application of the theory to experimental material is illustrated.     

EPR studies of a nonphotosynthetic mutant of Rhodospirillum rubrum

The EPR properties of P₈₇₀ and the primary electron acceptor in chromatophores from R. rubrum and a nonphotosynthetic mutant have been compared. Using steady-state illumination in the presence of various electron donors, it has been found that the primary acceptor in the mutant strain accumulates in the reduced state even under aerobic conditions while this behavior does not occur with the wild-type strain. The properties of the photoreduction of a bound iron-sulfur center which most likely functions in a substrate-linked dehydrogenase are the same in both strains. These results are discussed in terms of the requirement for a component (rhodoquinone) which regulates the redox state of the primary electron acceptor during normal photosynthetic growth but is not required during dark aerobic growth.     

Three new alpha-chains of collagen from a non-basement membrane source

Three new collagen α chains have been isolated from synovial membrane, gingiva and skin. Two of these have a similar chromatographic behaviour to the α[A] and α[B] chains described by Burgeson $\text{et al}$. (4) from a foetal basement membrane source but have been separated from another contaminating α chain, α[C]. The α[A] and α[B] chains are present in approximately equal amounts. They contain no detectable 3-hydroxyproline, are highly glycosylated and all sugar residues are present as the disaccharides. The percentage of hydroxylation of the lysine is of the order of 70%. Only a third of these hydroxylysine residues are glycosylated. The significance of these peptides, present in a tissue substantially free of basement membrane, is discussed.     

Accumulation of a metallo-carboxypeptidase inhibitor in leaves of wounded potato plants

Mechanical wounding of young potato plants induces over a two fold increase in inhibitory activity against the bovine pancreatic metalloexopeptidase carboxypeptidase A. This increase in inhibitory activity in both wounded and unwounded leaves parallels the increases of two inhibitors of bovine serine endopeptidases, trypsin and chymotrypsin. This suggests that the Proteinase Inhibitor Inducing Factor is regulating the synthesis and accumulation of inhibitors of two different mechanistic classes of proteases found in animals and microorganisms. These increases in antiproteolytic activities due to wounding support the hypothesis that this response is part of a defense mechanism directed against plant pests.     

Vasopressin affects the behavior of rats in a positively-rewarded discrimination task

The effects of subcutaneous injections of vasopressin were investigated in a study utilizing 72 male Long-Evans rats trained in an appetitive black-white discrimination T-maze task. Animals which were reinforced for choosing the black goal arm demonstrated prolonged extinction if they received vasopressin prior to daily extinction sessions. This effect was not observed in animals reinforced for choosing the white goal arm. Prolonged extinction was not found in animals which received vasopresson only during acquisition or in control animals which received saline. Speed and activity scores did not differentiate the groups. These results demonstrate that vasopressin can affect the behavior of rats on a positively-reinforced task.     

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta-O-acetyl-α-maltosyl fluoride, the structure of which was assigned by¹⁹F-and¹H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/v vs 1/S; reaction constants for crystalline, sweet-potato enzyme were K_m 3.6 mM and V_max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d-glucose residues.     

Detachment variants of Chinese hamster cells : Hyaluronic acid as a modulator of cell detachment

Variants of the Chinese hamster cell line CHO have been isolated and characterized with respect to attachment and trypsin- or EGTA-mediated detachment kinetics, cell morphologies, and the complex carbohydrates (labeled with [³H]glucosamine) of the cell surface. The variant which was more readily detached from the substratum exhibited a more rounded cell shape and had three times more label as hyaluronic acid on the cell surface than the parental cell. The slowly detaching variant had a morphology similar to the parental cell but only half the radioactivity ascribable to hyaluronic acid. Endogenous levels of cAMP were unaltered in the variants. Exogenous dbcAMP caused the cells to elongate and flatten but did not alter the characteristic detachment kinetics. The role of hyaluronic acid as a modulator of the cell substratum interface is discussed.     

Inhibition of a nuclease contaminant in the commercial preparations of Escherichia coli alkaline phosphatase.

Escherichia coli alkaline phosphatase is a valuable reagent for removal of terminal phosphate from both ribo-and deoxyribo-oligonucleotides or from restriction enzyme fragments of DNA. Some commercial preparations of this enzyme were found to be contaminated with nucleases which could degrade both DNA and RNA. These contaminating nucleases can be completely eliminated by carrying out the enzymic reaction in the presence of 0.1-1% sodium dodecyl sulfate without any loss of phosphatase activity. This report has immediate application in the sequence analysis of DNA or RNA.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.     

Biochemical studies on a low molecular weight antiplasmin of human blood.

We previously reported a low molecular weight antiplasmin present in human platelets and plasma. This material as isolated by ultrafiltration was heterogeneous. The fraction showing maximum inhibition has been further purified by countercurrent distribution. The N-terminal residue of this purified inhibitor is alanine. Amino acid analysis showed glycine to be the predominant residue, and no basic amino acid was detected. Kinetic studies suggested a competitive inhibition of plasmin via enzyme-inhibitor complex formation. Carbohydrate and phosphate could not be detected. These data were similar for both platelet antiplasmin and serum antiplasmin. The relationship between the platelet and the serum inhibitor remains unclear. This low molecular weight antiplasmin may contribute to the consolidation of the thrombus.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Identification of the small subunit of ribulose 1,5-bisphosphate carboxylase as a product of wheat leaf cytoplasmic ribosomes.

Polyribosomes from greening wheat seedlings (Triticum vulgaris) were allowed to incorporate [³H]leucine into proteins in the presence of a wheat germ supernatant fraction under conditions permitting the completion and release of polypeptides by cytoplasmic polyribosomes. The released proteins were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Discrete proteins, as well as a variety of poorly resolved proteins, were observed to have been labeled. The molecular weight distribution of the labeled proteins correlated well with the distribution of polyribosome size classes present in the samples. Neither the large nor the small subunit of ribulosebisphosphate carboxylase were detected as labeled peaks by this procedure.Immune precipitates formed by the addition of carrier small subunit, detergent, and anti-small subunit serum to the released proteins contained a substantial proportion of nonspecifically precipitated material resembling the population of released proteins, but they also contained two discrete peaks not resolved previously, one having mobility slightly faster than the light chain of immunoglobulin (20,000 daltons) and the other having mobility identical to that of small subunit carrier (ca. 12,000 daltons). Samples containing the latter material were shown to contain labeled tryptic peptides corresponding to those of the small subunit carrier. The results establish that the small subunit of ribulose bisphosphate carboxylase is the product of a small proportion of cytoplasmic polyribosomes during greening of etiolated wheat seedlings.     

The substrate analog bromopyruvate as a substrate, an inhibitor and an alkylating agent of malic enzyme of pigeon liver

Bromopyruvate is an alkylating agent of pigeon liver malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.40). It combines first with the enzyme to give an enzyme-bromopyruvate complex, then reacts with a proximal -SH group, resulting in the formation of a pyruvate derivative. Bromopyruvate is also a substrate for the reductase partial reaction, and a non-competitive inhibitor of L-malate in the overall oxidative decarboxylase reaction catalyzed by this enzyme. Modification of the -SH group by this compound is accompanied by concomitant loss of both oxidative decarboxylase activity and reductase activity on bromopyruvate. Inactivation of the overall activity is partially prevented by NADP⁺ or NADPH, singly or in combination with L-malate.     

Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate.

This report describes the preparation of a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate and its use in a sensitive fluorometric assay of neuraminidase (EC 3.2.1.18) from Vibrio cholerae, cultured fibroblasts, and human leucocytes. V. cholerae neuraminidase showed maximum activity at pH 4.6 and an apparent K_m of 1.5 mm and was activated by CaCl₂ and inhibited by ethylenediaminetetraacetate, NaCl, and N-acetylneuraminic acid. The inhibition by N-acetylneuraminic acid was competitive (K_i = 6.1 mm). Cultured fibroblast and leucocyte neuraminidases showed maximum activity between pH 4.2 and 4.4 and apparent K_m values of 0.13 and 0.22 mm, respectively. Neuraminidase activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III.