Preparation and characterization of tetrasaccharides from beef-lung heparin

Partial N-desulfation of beef-lung heparin prior to degradative deamination with butyl nitrite and reduction with sodium borotritide yielded many large fragments. From these, a tetrasaccharide tetra-O-sulfate (II-4NH; 8% yield from heparin) and a mixture of tetrasaccharide tri-O-sulfates (II-3NHh; 6% yield) were isolated by sequential chromatography on Sephadex G-25 and DEAE-Sephadex. For these and the other tetrasaccharide preparations, the radioactive disaccharides produced by deamination, with and without subsequent relabelling with sodium borotritide, have been quantitatively determined by the methodology described in the preceding paper. In most cases, the results permit a unique reconstruction of the relative proportions of monosaccharide components and of their sequences in the compounds present. Tetrasaccharide II-4NH appeared homogeneous and has the structure (IdoA-SO₄)(GN-O-SO₄)(IdoA-SO₄)(anhMan-SO₄). In tetrasaccharide preparation II-3NHh, the preponderant species (57%) lacks ester sulfate at the terminal l-iduronic residue in the structure just mentioned, and five other species are present. By treatment of the tetra-O-sulfate with mild acid, tetrasaccharide preparations with 3, 2, 1, and no ester sulfate were produced and could be isolated. The isomeric tetrasaccharide tri-O-sulfate species have been partially resolved. Composition and sequence data are given for all of the preparations. The resolution of numerous small fractions suggests minor irregularities in the fine structure of heparin. Ion-exchange electrophoresis was applied to the acidic oligosaccharides and was found to be a useful technique.     

Some physical and chemical properties of trypsin-digested nucleoprotein

A DNA-peptide complex that is soluble in 0.2m-sodium chloride can be prepared by trypsin digestion of calf thymus nucleoprotein. The trypsin-digested nucleoprotein molecule contains about 70% of DNA and 30% of peptides by weight, and consists of one DNA molecule associated with arginine-rich peptides. A series of trypsin-digested nucleoprotein preparations differing only in molecular weight were prepared by blending. The intrinsic viscosity and average sedimentation coefficient were determined for each of these preparations. Then the DNA was isolated from each preparation and the hydrodynamic measurements were repeated on the DNA. From a comparison of these results it was concluded that the presence of the complex-forming peptides causes a large decrease in intrinsic viscosity of the DNA and an increase in sedimentation coefficient. In addition, the hydrodynamic data indicate that the DNA-peptide complex behaves like a coil in solution but is more compact than the same length of DNA. The ;melting' profiles, streptomycin precipitation curves and maximum viscosities obtained with ethidium bromide binding for the trypsin-digested nucleoprotein are similar to those of purified DNA, and markedly different from those of undigested nucleoprotein. These findings suggest that the peptides are not strongly associated with the DNA, and that secondary valency forces are involved in the binding.     

Phenylalanine 4-monooxygenase from bovine and rat liver: Some physical and chemical properties

Phenylalanine 4-monooxygenase was purified from bovine liver using a modification of the procedure developed for the rat liver enzyme (Shiman, R., Gray, D. W., and Pater, A. 1979. J. Biol. Chem. 254:11300–11306). The enzyme preparation appeared essentially homogeneous on polyacrylamide gel electrophoresis under non-denaturing conditions. Electrophoresis in the presence of dodecyl sulfate revealed that about 95% of the protein had a mobility, corresponding to M_r=51,000. The remaining 5% was recovered in two minor bands corresponding to M_r of about 35,000 and 15,000 and is likely to result from limited proteolysis of the native enzyme with dissociation of the fragments on denaturation by detergent. The enzyme comigrated with the rat liver enzyme on polyacrylamide gel electrophoresis in both systems studied. No significant difference was observed between the amino acid composition of the bovine and rat liver enzyme, in the reactivity of their sulfhydryl groups or in their iron content (i.e. 1.5–3.0 iron atoms per peptide chain of M_r=50,000). Both enzymes contained less than 0.01 copper atom per peptide chain. The enzymes were inhibited in a similar manner by the chelator bathophenanthroline disulfonate (selective for iron and copper), but not by bathocuproine disulfonate (specific for copper). The results indicate that the bovine and rat liver enzymes are closely similar and that iron, but not copper, is essential for enzyme activity. High performance size-exclusion liquid chromatography revealed that both native enzymes exist in different oligomeric forms, but further studies are required to understand the physicochemical basis for this phenomenon.Abbreviations Bathophenanthroline 4,7-diphenyl-1, 10-phenanthroline - bathocuproine 2,9-dimethyl-4,7-diphenyl-1, 10-phenanthroline - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - dansyl 1-dimethylaminonaphthalene-5-sulfonyl - DMPH₄ 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine - M_r relative molecular mass     

Some enzymic activities and chemical properties of the mesosomes and cytoplasmic membranes of Bacillus licheniformis 6346

1. The liberation of the vesicles of the mesosomes from protoplasts of Bacillus licheniformis strain 6346 is controlled by the Mg(2+) concentration present during the removal of the walls by lysozyme. 2. The functioning of the cytoplasmic membranes is also critically controlled by the Mg(2+) concentration. 3. The isolated mesosomes and the cytoplasmic membranes differ in their enzymic activities. Both succinate dehydrogenase and NADH oxidase activities are very low or absent from the mesosomes. 4. The cytoplasmic membranes can also be separated into materials of different density. 5. Distribution of the membranes between these fractions is controlled by the Mg(2+) concentration and the growth conditions of the microorganisms.     

Some physical properties of paramyosin from earthworms

Paramyosin was prepared from earthworms (Lumbricus terrestris) by two different methods that have been used in the past. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the older method yields slightly degraded material (mostly β- and γ-paramyosin) while the newer method yields essentially intact, i.e., α-, paramyosin. Physical studies, particularly circular dichroism, light scattering, and sedimentation velocity show that the native molecule is a double α-helical coiled coil of molecular weight 200,000, length 1200 Å, and diameter 20 Å. These properties are the same as reported previously for molluscan paramyosin. Also like clam paramyosin, the worm protein molecule loses its helix content and dissociates into its two constituent polypeptide chains upon exposure to sufficient concentration of Gdn-HCl. Furthermore, the same partially denatured states can be reached from either native or completely denatured proteins, indicating that they are all equilibrium states. However, the Gdn · HCl-induced denaturation profile for the worm paramyosin is quite different from the clam. The helix content of worm paramyosin diminishes monophasically with increasing concentration of Gdn-HCl, showing that the molecule does not possess a region of special stability such as its clam analog boasts. This conclusion is supported by experiments on papain digestion of worm paramyosin, wherein no resistent core is seen.     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.