Occurrence of N-malonyl-D-alanine in pea seedlings

One of the ninhydrin-negative alanine conjugates isolated from pea seedlings was identified as $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$.The identification of this conjugate was carried out by a comparison of its gas-liquid chromatographic and mass spectrometric properties, and its nuclear magnetic resonance and infrared spectra with those of synthetic $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$. The alanine in the conjugate was shown to be present as the D-isomer by enzymatic and chromatographic analyses.     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.     

Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-³H]cytochalasin B and transport of $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-[}{}^{14}\text{C]glucose}$ into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a K_D of 2.86 · 10^?7 M, while the low-affinity site had a K_D of 1.13 · 10^?5M. Sugar transport was monitored by $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-}\text{glucose}$ uptake and it was found that cytochalasin B (10^?5M) drastically inhibited transport. However, D-glucose (10^?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.     

1-Aminoglycosides, a new class of specific inhibitors of glycosidases

1-Aminoglycosides represent a new class of specific and relatively potent inhibitors of glycosidases. These compounds are specific against those enzymes which act upon glycosides that correspond to glycone of the inhibitor. Thus α- and β-$\text{D}\text{=}$- are inhibited by $\text{D}\text{=}$-glucosidases but not by $\text{D}\text{=}$-galactosylamine and $\text{D}\text{=}$-mannosylamine. α- and $\text{D}\text{=}$-galactosidases are inhibited by $\text{D}\text{=}$-galactosylamine but not by the other two glycosylamines. $\text{D}\text{=}$-Mannosylamine inhibits mannosidase.     

Purification and characterization of insect cathepsin D

We purified a proteolytic enzyme from pupae of the blowfly Aldrichina grahami. The purification procedure consisted of fractionation with ammonium sulfate, ethanol treatment, affinity chromatography on Con $\text{A-}\text{Sepharose}$, and ion exchange chromatography on CM-Sepharose CL-6B. The purified enzyme was very labile. The lability was reduced by the use of MES buffer containing 10% ethanol, which enabled us to purify the enzyme to homogeneity. The lower the polarity of the alcohol added, the more stable the enzyme became. The enzyme had a molecular weight of 41,000, an optimum pH of 3.5, high susceptibility to pepstatin and two pI forms of 5.4 and 6.2. This enzyme resembled cathepsin $\text{D}$, a lysosomal proteinase.     

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.     

Purification of penicillin-insensitive DD-endopeptidase, a new cell wall peptidoglycan-hydrolyzing enzyme in Escherichia coli, and its inhibition by deoxyribonucleic acids.

Activity of a penicillin-insensitive $\text{DD}$-endopeptidase that splits the $\text{D}$-alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of $\text{Escherichia coli}$. The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments.     

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D[U-¹⁴C]mannose or $\text{N-}\text{acetyl}\text{-}\text{D}\text{-[1-14}\text{C}\text{]-}\text{glucosamine}$). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver α-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electropresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{D}\text{-}\text{GlcNAc}\text{(I), α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)- α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (II), and $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)-α-}\text{D}\text{-}\text{Man}\text{- (1 → 2)-α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean α-mannosidase gave D-mannose and a disaccharide (containing D-mannose and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$). Thus the three main compounds observed in the fibroblast from patients with mannosidosis are most probably identical to the oligosaccharides I–III.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

Occurrence of cholecalciferol in the calcinogenic plant Trisetum flavescens.

The grass Trisetum flavescens causes severe calcification of soft tissues upon ingestion by various species, which has been ascribed by others to a 1,25(OH)₂ vitamin D₃-like activity.By a special purification procedure involving high pressure liquid chromatography and continuous biological testing the active principle was purified. By means of $\text{GC}\text{MS}$ it was identified as cholecalciferol, being present in a concentration of about 0.1 ppm in the lyophylized plant dry matter. 1,25(OH)₂ vitamin D₃ or other metabolites of vitamin D₃ were not present. Since such low concentrations could hardly explain the calcinosis observed, a more active “bound form” of vitamin D₃ may be present in Trisetum flavescens.     

A new category of ovulation inhibitors: linear LH-RH analogues having more than ten residues.

A linear analogue of the luteinizing hormone-releasing hormone, longer than a decapeptide, is described for the first time, which is equivalent in potency to the best known inhibitors of ovulation, and which constitutes an important new lead to the design of inhibitors of even greater potency. At a dosage of 200 μg/rat, the undecapeptide [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH caused 100% inhibition of ovulation. The related analogues, [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH and [(Gly-Pro)¹, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH, were less active, $\text{in}\text{vivo}$. All of these undecapeptides inhibited the action of 0.6 ng/ml of LH-RH by greater than 50% at the very low level of 10 ng/ml.     

The interaction of Ricinus communis hemagglutinin with polysaccharides and low molecular weight carbohydrates

The hemagglutinin from the castor bean (Ricinus communis) shows a precipitin-like like reaction with a series of branched galactomannas, dependent on their galactose: mannose ratio. Charged and neutral linear galactants fail to co-precipitate with the protein. Hapten inhibition of the turbidimetrically assayed hemagglutinin-Lucerne seed galactomannan system incidates that simple sugars such as D-galactose, D-fucose and L-arabinose bind to the protein. Of the glycosides tested, methyl β-D-galactopyranoside is a better inhibitor than the corresponding α-another. $\text{p-}\text{Nitrophenyl}\text{-2-}\text{acetamido}\text{-2-}\text{deoxy}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$ is about 10 tiems less effective than $\text{p-}\text{nitrophenyl}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$, the best inhibitor tested. Equilibrium dialysis data obatined with the latter ligand are consistent with a protein containing two identical and independent binding sites with an intrinsic association constant equal to 1.65 ? 10⁴ l/mole at 25 °C.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.     

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.