The ilvEDA operon of Escherichia coli K12 encodes only one valine-alpha-ketoglutarate transaminase activity.

Transaminase B of $\text{E. coli}$ K12 was purified to apparent homogeneity as measured by SDS acrylamide gel electrophoresis, immunoelectrophoresis, and amino terminal sequence analysis. The valine- and isoleucine-α-ketoglutarate dependent transaminase activities of pure enzyme as well as crude extracts were characterized by immunologic and kinetic methods. The data disprove the existence of a separate valine-α-ketoglutarate transaminase within the $\text{ilvEDA}$ operon.     

Requirement of different ionic concentrations for optimal translation of α and β globin mRNA in Krebs II ascites cell-free system

The translation of rabbit α globin mRNA in a Krebs II ascites cellfree system was more dependent upon the K⁺ concentration than rabbit β globin mRNA. The optimal KCl concentration was approximately 70 mM for the synthesis of the α chain and between 80 and 90 mM for that of the β chain. With CH₃ CO₂K the optimum concentration for α chain synthesis was also 70 mM but the optimum for the β chain synthesis was not sharp any more and ranged from 70 mM to over 110 mM. In the range of the optimal Mg²⁺ concentration for the α and β globin chain synthesis the $\text{α}\text{β}$ ratio decreased when the Mg²⁺ concentration increased. In the presence of DTT and EDTA the optimal KCl concentration for both α and β globin chain synthesis decreased.     

Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

A general NMR method for the assay of racemase activity with optically active or optically inactive substrates

A rapid and sensitive method is described for determining the activity of alanine racemase (5.1.1.1) with its substrates. Alanine racemase from Bacillus subtilis catalyzes the exchange of the α hydrogen of l and d alanine, a racemic mixture of alanine as well as both the α hydrogens of glycine with D₂O. The rate of exchange which can be followed by NMR spectroscopy is proportional to the enzyme concentration. This assay has been used to test a wide variety of possible substrates and thus determine the specificity of the enzyme.     

The crystal structure of galactaric acid (mucic acid) at −147°: An unusually dense,hydrogen-bonded structure

Galactaric acid, C₆H₁₀O₈, (CAS Reg. No. 526-99-8), is triclinic, P$\text{1}$, with cell dimensions at ?147° [and 20°], a = 4.900(1) [4.918(1)], b = 5.728(1) [5.816(1)], c = 6.784(1) [6.849(1)] Å, α = 92.32(2) [92.31(2)], β = 93.74(2) [94.16(2)], γ = 93.08(2) [93.49(2)]°, V = 189.5 ų, Z = 1, D_x = 1.831 [1.800], D_m = [1.790] g.cm^?3, molecular symmetry $\text{I}$. The structure was solved by the direct method, MULTAN, and refined to R = 0.034, R_w = 0.039 for 787 reflections with F_Obs > 3σ(F_obs). The crystal structure has a system of strong, intermolecular hydrogen-bonds, which accounts for the high crystal density and low solubility in water.     

Chemical modification of opiate receptors with ethoxyformic anhydride and photo-oxidation: evidence for essential histidyl residues

In the presence of $\text{D}\text{=}$-carnitine significant decarboxylation of 2-oxoglutarate occurs with γ-butyrobetaine hydroxylase (EC 1.14.11.1) both from $\text{Pseudomonas}$ sp AK 1 and from human kidney. No product was formed from carnitine when $\text{D}\text{=}\text{L}\text{=}$-carnitine was incubated with either enzyme but succinate was formed in 1:1 stoichiometry to decarboxylation using $\text{D}\text{=}$-carnitine and the human enzyme. $\text{L}\text{=}$-Carnitine is also an uncoupler for the human enzyme. There is no significant decarboxylation of 2-oxoglutarate in the absence of a substrate, but during normal catalysis in the presence of γ-butyrobetaine the formation of CO₂ from 2-oxoglutarate exceeds carnitine formation with 20% for the human enzyme.     

The use of affinity elution from Blue Dextran Sepharose by yeast tRNA2Val in the complete purification of the cytoplasmic valyl-tRNA synthetase from Euglena gracilis

Two distinct monomers, α and β participate in the structures of diffe$\text{r}$ ent oligomers of $\text{Neurospora crassa}$ glutamine synthetase (EC 6. 3. 1. 2). In ammonium-limited cultures a tetrameric form composed mainly of α monomers was found. In excess of nitrogen an octameric form composed mainly from β monomers is the predominant oligomeric state. The presence of both monomers was observed in intermediate oligomeric forms.     

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.     

Metabolism of L-beta-lysine in a Pseudomonas: conversion of 6-N-acetyl-L-beta-lysine to 3-keto-6-acetamidohexanoate and of 4-aminobutyrate to succinic semialdehyde by different transaminases.

Some kinetic properties of two new species of transaminase found in extracts of a β-lysine-utilizing Pseudomonas are reported. Transaminase A catalyzes transamination between 6-N-acetyl-l-β-lysine (3-amino-6-acetamidohexanoate) and α-ketoglutarate to form 3-keto-6-acetamidohexanoate and glutamate. Transaminase B catalyzes a reaction between 4-aminobutyrate and pyruvate to form succinic semialdehyde and alanine. The formation of both transaminases is induced by growth of the bacteria on l-β-lysine, although transaminase B is also produced in the absence of this substrate. Transaminase A requires pyridoxal phosphate for activity. The β-keto acid formed from acetyl-β-lysine by transaminase A has been purified and characterized by decarboxylation, conversion to a formazan, reduction to a stable β-hydroxy acid, and conversion of the latter to its methyl ester. Transaminase B, unlike previously reported transaminases utilizing 4-aminobutyrate, cannot use α-ketoglutarate as an amino group acceptor. This enzyme is not stimulated by addition of pyridoxal phosphate, but is inhibited by hydroxylamine or cyanide. Both transaminases appear to function in the main pathway of β-lysine degradation.     

Characterization of the Ca2+-regulatory complex of chick embryonic muscles: Polymorphism of tropomyosin in adult and embryonic fibers

The Ca²⁺-regulatory tropomyosin-troponin complex was purified from chick embryonic muscles by a combination of DEAE-cellulose chromatography and (NH₄)₂SO₄ fractionation. The embryonic complex was very similar to that obtained from adult chicken muscles with respect to stoichiometry of components and biological activity. Tropomyosin of embryonic skeletal muscles contains both α and β subunits, the β form being the major species. In the adult stage the β form is decreased with a concomitant increase in the α form. These results indicate that i) the Ca²⁺-regulatory proteins are not deficient in early embryonic muscles as previously thought (Hitchcock, S.E., Develop. Biol. $\text{23}$, 399, 1970), and ii) different structural genes coding for tropomyosin subunits are expressed differentially in embryonic and adult muscle fibers.     

Relative biological activity of certain prostaglandins and their enantiomers

A series of mirror image ($\text{ent}$) forms of prostaglandins F₂ and E₂ have been compared for potency in a hamster antifertility test. In the PGF₂ series, $\text{ent}$-compounds surveyed had less potency than corresponding natural structures. For the PGE₂ series, 11α-(15S)-ent-PGE₂ methyl ester was 10-fold more potent than PGE₂. Altering the C-9 hydroxy configuration in the PGF₂ series from the natural α to β decreased potency dramatically for compounds tested.     

Solvent and substrate deuterium isotope effects on a transamination reaction catalyzed by pig heart aspartate aminotransferase.

Transamination of erythro-β-hydroxy-l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in $\text{H}{}_2\text{O}\text{D}{}_2\text{O}$. The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D₂O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Effect of light and darkness on pineal hydroxyindole-O-methyl transferase (HIOMT) in Japanese quail

The optimal incubation temperature for assay of the melatonin-forming enzyme, hydroxyindole-O-methyl transferase (HIOMT), of the Japanese quail pineal, was determined to be 47° C. In adult quail, HIOMT activity was high in continuous light and in the light phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$, and was low in continuous darkness and in the dark phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$. Age of the bird and length of the lighting treatment seemed to be important factors in demonstrating this effect of light and darkness on HIOMT activity.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Crystallographic data for lysozyme from the egg white of the Embden goose

Two crystal forms of lysozyme from the egg white of the embden goose (Anser anser) have been obtained, both of which are suitable for X-ray diffraction analysis. The monoclinic form has space group P2₁ with cell dimensions $\text{a = 38.3}\text{A}\text{?}\text{, b = 65.7}\text{A}\text{?}\text{, c = 45.2}\text{a}\text{?}$, β = 116 ° and the triclinic form (space group P1) has cell dimensions of $\text{a = 39.9}\text{A}\text{?}\text{, b = 42.2}\text{A}\text{?}\text{, c = 57.9}\text{A}\text{?}$ and α = 98.8 °, β = 102.5 °, γ = 90.5 °.