Comparative Zone Electrophoresis of Enzymes of Pseudomonas solanacearum and Pseudomonas cepacia

The technique of starch-gel electrophoresis with specific staining for a series of enzymes was used to compare 21 Pseudomonas strains representing both P. cepacia and P. solanacearum. These experiments produced no evidence for close similarity of the two species. Twelve strains of P. solanacearum were compared by means of data obtained from nine different enzymes, and the data indicate that these strains belong in two biotypes. Except for the assignment of two strains, these groups are the same as the two major groups previously derived from nutritional properties and from deoxyribonucleic acid hybridization experiments. Eleven enzymes were available for comparisons of the P. cepacia strains. Eight of these strains form a homogeneous group, but the last strain, number 249, differs considerably from the other representatives of the species.     

Altered proteins with triosephosphate isomerase activity in suppressor-containing strains of Bacillus subtilis

Suppressor mutations in Bacillus subtilis cause the synthesis of a new protein with the enzymatic activity of l-leucine dehydrogenase and two groups of new proteins with the activity of triosephosphate isomerase. The new isoenzymes of triosephosphate isomerase are separable by zone electrophoresis and differ among themselves in elution behavior upon gel permeation chromatography. One group has an apparent average molecular weight of 120,000 to 135,000, which is more than twice that of the wild-type enzyme. Another group appears to be even higher in molecular weight. These data are consistent with the working hypothesis that the new isoenzymes are produced by extension of growing polypeptide chains through one or more chain-terminating triplets, although other mechanisms resulting in alteration of shapes, charges, or associations of the enzymes are not excluded.     

Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants

1. ATP sulphurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PP(i)-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PP(i)-ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and K(m)(selenate)/K(m)(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PP(i)-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PP(i) is the first product released. 4. Synthesis of adenosine 5'-[(35)S]sulphatophosphate from [(35)S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg(2+)-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [(75)Se]selenate as substrate could not be demonstrated.     

An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids

A sensitive electron-capture gas-liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F(2alpha) was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ;on-column' as the silylated product. The method was successfully applied to the determination of prostaglandin F(2alpha) in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas-liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F(2alpha) in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F(2alpha). The electron-capture gas-liquid-chromatographic assay is compared with the radioimmunoassay and the gas-liquid-chromatographic-mass-spectrometry assay for the determination of prostaglandin F(2alpha).     

Cyclic metabolic pathway of a butylated hydroxytoluene by rat liver microsomal fractions

A cyclic metabolic pathway was obtained when 3,5-di-t-butyl-4-hydroxytoluene (BHT) was incubated with a rat liver microsomal preparation. The pathway is as follows: BHT --> 4-hydroperoxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-OOH) --> 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-3(0)OH) --> BHT. This metabolic pathway suggests that antioxidants such as BHT owe their high efficacy, at least in part, to their metabolic regeneration.     

The mineralization and hardness of the radular teeth of the limpet Patella vulgata L.

Summary A study of the Patella vulgata radula has been made using: the scanning electron microscope in its normal and compositional contrast modes of operation, the electron microprobe analyser, ion etching with argon ions and microhardness testing.Only iron, silicon and small amounts of sulphur were detected in the radula. The teeth can be subdivided into a cusp, a junctional area where the cusp is joined to the base, and the base which is embedded in the radular membrane. From a study of longitudinal vertical and transverse sections of the mature teeth it was found that the cusp could be subdivided into a posterior iron-rich area (44–51% Fe, 1–6% Si) and an anterior silicon-rich area (22–30% Fe, 27–32% Si). The junctional zone consisted of a poorly mineralised layer at its border with the cusp and an iron-rich layer where it joined the base. The upper part of the base (5% Fe, 16% Si) could be clearly differentiated from the silicon-rich anterior and lower parts of the base (3–4% Fe, 28–35% Si). No minerals were detected in the membrane. The changes in the mineral content of the teeth cusps along the length of the radula were studied. Iron appeared in the cusps at the 25th row and the concentration increased to 28% at the 50th row. The iron was here evenly distributed throughout the cusp. Silicon appeared in the anterior part of the cusp at the 50th row and as it increased in concentration so the iron was displaced, and at the same time the concentration of iron increased in the posterior part of the cusp. Mineralization appeared to be complete by the 150th row.The teeth cusps appear to consist of 800 Å fibres grouped into 1 thick bundles and the tooth appears to be covered by a thin enamel-like layer. It is suggested that the fibres contain the silicon-rich phase and the matrix the iron-rich phase.The significance of the arrangement of the fibres and the distribution of the minerals are discussed with relation to the function of the teeth.We wish to thank Mr. A. Rees and Mr. A. Davies for their technical assistance; Prof. Lewis and Dr. James for the use of the Electron Microprobe; and the S.R.C. for their financial support.