Enzymatic synthesis of acrylamide: a success story not yet over.

The application of enzymatic transformations is attracting increasing attention. Until recently, such an approach has generally been confined to producing fine chemicals difficult to obtain through conventional chemical methods. Microbial nitrile hydratase (NHase) has now been applied to the industrial, kiloton-scale production of the important chemical commodity acrylamide. Recent progress in understanding microbial nitrile metabolism at both the gene and protein levels is permitting improvement of the acrylamide production process.     

Purification of achatin-I from the atria of the African giant snail, Achatina fulica, and its possible function.

Achatin-I previously purified from the ganglia of the African giant snail Achatina fulica was isolated from the atria of this snail. Achatin-I appeared to enhance the cardiac activity in two ways; centrally this peptide increased impulse frequency and produced spike broadening of the identified heart excitatory neuron, PON, and peripherally it enhanced amplitude and frequency of the heart beat. Achatin-I showed excitatory actions not only on the heart but on several other muscles.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Binding of staphylococcal cell surface polysaccharide to human fibrinogen

The interaction between the binding site of a polysaccharide (called compact colony forming active substance (CCFAS)), obtained from the cell surface of a strain of Staphylococcus, and human fibrinogen (HF) was investigated. The CCFAS was found to bind specifically to both the B beta and gamma chains of HF at pH 7.0 and 8.0, and the A alpha chain at pH 5.0. The binding of CCFAS with fibrinogen fragments obtained by digestion with plasmin were also investigated. Fragments with Mr of 55,000, 24,000, and 19,000 were the major bands precipitated by CCFAS at pH 7.0 and 8.0. Fragments with Mr of 85,000 and 75,000 bound to CCFAS at pH 5.0. Binding of CCFAS (7 micrograms) with fibrinogen could be inhibited by 1.2 micrograms of B beta chain and 1.5 micrograms gamma chain at alkaline pH or 6.2 micrograms of the A alpha chain at pH 5.0. CCFAS was, therefore, assumed to be specifically bonded with HF molecules, in the alkaline range at least, resulting in compact colony forming activity in serum soft agar and paracoagulation.     

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.     

Anti-platelet aggregating and disaggregating activities of 6,9-methano PGI2

Anti-platelet aggregating and disaggregating activities of the chemically stable 6,9-methano prostaglandin I₂ (6,9-methano PGI₂) were investigated. 6,9-Methano PGI₂ inhibited ADP-induced platelet aggregation in PRP from humans, rabbits and rats. 6,9-Methano PGI₂ also inhibited rabbit platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and 11,9-epoxy-methano PGH₂. Antiaggregating activities of 6,9-methano PGI₂ were 0.3 to 2.0 times greater than those of PGE₁. 6,9-Methano PGI₂ facilitated platelet disaggregation in a dose related manner. Antiaggregating and disaggregating activities of 6,9-methano PGI₂ were markedly enhanced by incubation with the phosphodiesterase inhibitor, theophylline.     

Ca2+ regulated modulator protein interacting agents: inhibition of Ca2+-Mg2+-ATPase of human erythrocyte ghost.

Agents such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and its derivatives, chlorpromazine and amitriptyline that interact with calcium-regulated modulator protein were found to inhibit not only Ca²⁺ dependent cyclic nucleotide phosphodiesterase but also Ca²⁺-Mg²⁺-ATPase of human erythrocyte ghosts. I₅₀ values of modulator interacting agents for testis modulator-activated, brain modulator-activated and erythrocyte modulator-activated-ATPase are indistinguishable. However, I₅₀ of W-7 for troponin C-activated-ATPase is lower than that for modulator-activated ATPase. The specificity of these agents toward modulator-related enzyme reaction is also shown by the negative effect on modulator-unrelated enzyme system such as erythrocyte ghost protein kinase and Mg²⁺-ATPase. These agents provide a useful tool for elucidating the physiological role of modulator.     

Insulin release and metabolism of calcium, adenine and adenosine 3',5'-cyclic monophosphate.

In order to assess further the mechanisms involved in insulin release, we prelabeled rat pancreatic islets of Langerhans by incubating either 45Ca or [2-3H]adenine. When prelabeled islets were perfused with a glucose-free medium (the experiment with 45Ca) and a medium containing 2.8 mM glucose (the experiment with [2-3H]adenine) respectively, a constant rate of efflux of the radioactivity was established by 30 min in each case. D-Glucose at 16.7 mM concentration elicited a rapid efflux of 45Ca and [2-3H]adenine derivatives ([3H]Ad) within 4 to 6 min after commencing the step-wise stimulation by glucose, concomitantly with insulin release. However, L-glucose and D-galactose littel stimulated both 45Ca and [3H]Ad release. Lanthanum chloride caused a burst peak of 45Ca release in the absence of glucose. A rapid efflux of 45Ca was caused by beta-D-glucose and D-glyceraldehyde to much lesser extent than by alpha-D-glucose. The slowly rising concentration of glucose at 0.1 mM/min of gradient level failed to elicit any rapid efflux of 45Ca or [3H]Ad, although insulin release occurred in accordance with an increase in glucose concentration. Even when the gradient of glucose concentration was raised to 0.7 mM/min, glucose failed to stimulate an efflux of [3H]Ad but the subsequent stimulation by 16.7 mM glucose caused a rapid efflux of [3H]Ad concomitantly with the release of insulin. No rapid efflux of 45Ca was observed under a slow-rise glucose stimulation until the gradient level of the glucose concentration was raised to 6.7 mM. Analysis of distribution of the radioactive adenine derivatives after incubation showed that the adenosine fraction had the highest radioactivity in the medium followed by the ATP, adenine and cAMP fraction in that order, and the ATP fraction had the highest radioactivity in the islet. The ratio of radioactivity in the cAMP fraction in the medium to the total count was the highest among all. On the basis of these results, it was suggested that the discharge of [3H]Ad and 45Ca might occur with the alteration of the membrane permeability induced by a rapid change of the glucose concentration, and that their discharge might perhaps link to the glucoreceptor mechanism directly controlling insulin release.     

R plasmic Rtsl-mediated production of extracellular deoxyribonuclease in Escherichia coli.

Escherichia coli strains harboring Rtsl were found to excrete extracellular deoxyribonuclease. The DNase activity was greater in cells with pTW2, a mutant from Rtsl.     

Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.