A. D. Larson

Purification and characterization of the extracellular proteinase of Serratia marcescens

The extracellular proteinase produced by a depressed strain of Serratia marcescens ATCC 25419 was purified and characterized. This produces more than 10-times the amount of extracellular proteinase produced by other strains of Serratia tested. The purified enzyme was prepared from the culture supernatant by (NH4)2SO4 fractionation and DEAE-cellulose chromatography. The purified enzyme has an so20,w of 3.95 and is a monomer of molecular weight 51,900. The proteinase has a broad pH optimum in the alkaline range with a maximum at pH 9.5. The enzyme will utilize a number of proteins as substrate. The products of digestion are primarily in the size range of tripeptides to hexapeptides. Peptides containing a sensitive bond and a minimum size of size amino acids are hydrolyzed. The proteinase is inhibited by chelating agents but unaffected by sulfhydryl or serine reagents and is devoid of esterase activity.

Purification and characterization of fumarase from Pseudomonas putida

Abstract Fumarase (fumarate hydratase EC 4.2.1.2.) from Pseudomonas putida was purified 790 times the specific activity of the crude extract. A molecular weight of 166,000 was obtained by sucrose density gradient centrifugation. Low concentrations of thiol inhibitors (10−4 m ) did not inhibit the enzyme, but preincubation of the enzyme with high concentrations (0.05 m ) of the inhibitors markedly decreased enzyme activity. The catalysis of the hydration of fumarate and dehydration of l -malate by fumarase was measured spectrophotometrically over a range of substrate concentrations of 2.5 × 10−4 −5 × 10−3 m for l -malate and 10−4 −10−3 m for fumarate. The studies were conducted over a pH range of 5.5–9.5 in 0.05 m sodium phosphate buffer at 30 °. The pH dependence of maximum initial velocity exhibited maxima at 6.9 with fumarate, and 8.1 with malate, as substrates. The same ionization constant values were demonstrated for the two essential groups in the free enzyme for either substrate which indicated that the same enzymatic site converts l -malate to fumarate and fumarate to l -malate. The ionization constants for the enzyme-substrate complex agree closely with the ones obtained for the porcine enzyme and indicate the mechanism of action of bacterial enzyme to be identical to the porcine enzyme. Michaelis constants and maximum initial velocity constants were decreased by increasing concentrations of phosphate buffer. It is thus possible that phosphate influences the ionization of groups in the active site of the enzyme or slightly changes the configuration of the site.

Preparation of intactAnaplasma marginale devoid of host cell antigens

A procedure for preparation ofAnaplasma marginale free of erythrocytic antigens is deseribed. The protocol employed heat-stable hemolysin produced byPseudomonas aeruginosa to lyse the erythrocytes and releaseA. marginale. Most of the contaminatng erythrocytic components were solubilized by enzymatic digestion. Final purification was accomplished by differential centrifugation. Electron microscopy and serological criteria established the absence of contaminating host cell antigens.

Purification, Characterization, and Quantitation of the Antigen Employed in the Immunodiffusion Test for Diagnosis of Equine Infectious Anemia

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.

Delayed lethal effect of 2,4-Dichlorophenoxyacetic acid on bacteria

tolerance of bacteria to 2, 4-dichlorophenoxyacetic acid (2, 4-D)was increased by continued culturing in the presence of sublethal concentrations of the herbicide. The latter authors reported that sublethal quantities of 2, 4-D did not effect cell size or the total cellular yield of Aerobacter aerogenes. They also presented convincing evidence that magnesium. deficiency in A. aerogenes did not mimic the action of sublethal quantities of 2, 4-D. Hart and Larson (3) reported on the effect of 2, 4-D on different metabolic types of bacteria and had observed that certain bacteria, particularly the Gram-negative facultatively anaerobic bacteria, showed a graded response to increasing concentrations of the herbicide. In further studies we have noted a delayed lethal effect and an effect on length of cells by 2, 4-D at concentrations which do not inhibit growth completely. The present report is concerned with these effects.

Purification of the Protein Employed in an Immunodiffusion Test to Diagnose Infectious Bovine Rhinotracheitis

The antigen used in an immunodiffusion test to diagnose infectious bovine rhinotracheitis has been purified by affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A So20,w of 0.749 was determined and a molecular weight of 8900 was calculated from sedimentation equilibrium analysis. The purified antigen formed precipitin lines of identity with crude diagnostic antigen. Purified antigen remained serologically active in the immunodiffusion test after lyophilization and subsequent reconstitution.