Michael Flashner

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

Abstract 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with Ki = 1.4 × 10−6 M and 4.8 × 10−5 M , respectively. The Km for the substrate, N-acetylneuraminlactose, is 1.0 × 10−3 M . These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.

Purification and properties of Arthrobacter neuraminidase

Neuraminidase (EC 3.2.1.18) from an Arthrobacter species was purified homogeneity by conventional procedures (yield approx. 1 mg/1) and was judged to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Gel electrofocusing of neuraminidase revealed 1 major band (85-90%), pI 5.35 +/- 0.05, and 6 minor bands, whose pI ranged from 5.25 to 5.70, and each of which had catalytic activity. Arthrobacter neuraminidase is a monomeric glycoprotein of molecular weight 88 000, has an apparent Km of 7.8-10(-4) M for N-acetylneuraminlactose, is insensitive to inhibition by N-acetylneuraminic acid, and is about 2% carbohydrate by weight. The amino acid composition as well as the galactosamine and glucosamine content was determined. The enzyme can hydrolyze (alpha, 2-3), (alpha, 2-6), (alpha, 2-8) linkages. The active size of the enzyme appears to be inaccessible since no inhibition was observed by reagents known to modify sulfhydryl, lysyl, carboxyl, histidinyl, and argininyl residues. In contrast, N-bromosuccinimide at a 60-fold molar ratio to enzyme, gave complete inhibition. These results suggest that a tryptophan residue is essential for catalysis.

Evidence for selective sulfhydryl reactivity in cytochalasin A--mediated bacterial inhibitions.

Abstract Cytochalasin A (CA) at 5 × 10 −5 M strongly inhibits glucose transport in Arthrobacter sialophilis . This effect and other bacteriostatic and metabolic inhibitions of gram-positive bacteria are not caused by the closely related congeners cytochalasin B or D. Inhibitions by CA are nullified by prior drug incubation with sulfhydryl compounds. It was also found that the characterized adduct of CA with β-mercaptoethanol is devoid of biological activity. N-ethylmaleimide, p-chloromercuribenzoate and ethacrynic acid (a known, liposoluble, sulfhydryl reactant) were each shown at 5 × 10 −5 M to be relatively ineffective in inhibiting D-glucose transport in A. sialophilus . These observations suggest that CA reacts at the molecular biological level in a site-specific manner.

Structure-activity relationships of cytochalasins in the differentiation of cytolytic T lymphocytes.

Four naturally occurring cytochalasins and three synthetic congeners have been studied for their effects on in vitro sensitization of murine lymphocytes to P815 mastocytoma. The relative order of effectiveness of these secondary fungal metabolites in inhibiting cytotoxic T cell development is as follows: cytochalasin D greater than cytochalasin E greater than cytochalasin A greater than cytochalasin B, 21,22- dihydrocytochalasin A greater than 7- acetylcytochalasin D. The 7,20 diacetylcytochalasin B derivative was inactive at the highest level tested (4 X 10(-6) M). Cytochalasin D is the most effective compound, producing at 5 X 10(-8) M a 50% inhibition of 51Cr release in a 4-hr cytolysis assay. This response pattern is in keeping with other test systems that implicate actin involvement, and underscores the contribution of an unsubstituted 7-hydroxyl drug function in receptor recognition. Inhibition produced by the cytochalasins is reversible if the compounds are removed from the tissue culture medium within the first 24 hr of a 4-day culture period. Delayed addition of cytochalasin D inhibits T cell development only within this first 24 hr of culture. These data suggest that the effects of cytochalasins are at an early step in the sensitization process, possibly antigen recognition.

Radioimmunoassay of Arthrobacter neuraminidase: applications to catalytic and taxonomic studies.

Abstract A radioimmunoassay capable of measuring nanogram quantities of Arthrobacter sialophilus neuraminidase was developed. Neuraminidases from several influenza viruses, Clostridium perfringens, Vibrio cholerae, Diplococcus pneumoniae , several group B streptococcal isolates, and human fibroblasts each failed to react with antisera raised in guinea pigs against the homogeneous A. sialophilus enzyme. Cross-reactivity was limited to neuraminidases from other Arthrobacter isolates and weakly, also with a Corynebacterium diphtheria enzyme preparation. Among the Arthrobacter -derived enzymes examined, that obtained from A. ureafaciens appeared nearly identical by this immunochemical criterion. Other designated Arthrobacter strains produced isofunctional proteins which exhibited distinct serological differences. The monospecific antibody also inhibited enzymatic activity of Arthrobacter neuraminidases. In the case of enzyme from A. sialophilus , inhibition by homologous antibody ranged from almost complete with Collocalia mucoid (a high-molecular-weight substrate) to none using sialyllactose. This substrate-dependent differential inhibition supports a steric model for inhibition of enzyme catalysis. Each of the cross-reacting Arthrobacter enzymes showed differential inhibition with sialyllactose, indicating variation in the positioning of one or more determinants with respect to their active sites. Further applications for this radioimmunoassay in relation to the use of neuraminidase in cell and molecular biology are discussed.

Rabbit muscle pyruvate kinase. Amino- and carboxyl-terminal studies

Amino-terminal analysis of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) failed to detect the presence of any free amino-terminal residues. Acetyl group analysis demonstrated the presence of between 3.7 and 4.0 mol of acetyl groups per mol of enzyme. The acetylated amino-terminal residue was isolated from pronase digests of the enzyme and identified as N-acetylserine. Quantitative recovery experiments indicated that all acetyl residues are found at the amino termini. Carboxyl-terminal analyses using the tritium exchange method suggested the presence of a blocked carboxyl-terminal residue, supporting previous hydrazinolysis and carboxypeptidase studies.

Photoaffinity labeling of plasma membrane receptors for cytochalasins in Ehrlich tumor cells

Treatment of purified Ehrlich ascites cell plasma membranes either with [3H]cytochalasin B or [3H]19-O-acetylchaetoglobosin A under photolytic conditions produced several radioactive polypeptides which were characterized by SDS-PAGE analyses. The major proteins so photolabeled were in the 60,000-80,000 Da range, with less labeling found in polypeptides smaller than 43,000 and greater than 90,000 Da. Immunofluorescent staining failed to identify the major photolabeled component as actin. It is concluded, in keeping with prior investigations using other cell types, that the predominant proteins photolabeled by cytochalasins are affiliated with the glucose-transport system.

Properties of Arthrobacter sialophilus Neuraminidase

Publisher Summary This chapter discusses the properties of arthrobacter sialophilus neuraminidase. It discusses the induction of an extracellular neuraminidase by a sialophilus by purification of A. sialophilus neuraminidase by conventional procedures and further analysis by various techniques such as disc gel electrophoresis. After purification, the properties of neuraminidase were investigated. The following cations, CA 2+ , Mg 2+ , Mn 2+ , and Co 2+ , at concentrations of 1 and 10 mM had no effect on neuraminidase activity whether included in the assay mixture or preincubated with the enzyme for 30 min at 37°C. EDTA tested under the same conditions also had no effect on neuraminidase activity. The chapter explains various properties of arthrobacter sialophilus neuraminidase.