Assay of 1L-myo-inositol-1-phosphatase using a fluorometric method.

1L-myo-Inositol-1-phosphatase, an enzyme purified from brain tissues, catalyzes the dephosphorylation of 1L-myo-inositol 1-phosphate. This enzyme has become the subject of intense research interest, since myo-inositol is needed for the resynthesis of phosphatidylinositol. We have developed a sensitive fluorometric assay for detecting the activity of 1L-myo-inositol-1-phosphatase. The assay is based on o-aminobenzoyl beta-glycerophosphate fluorescence, according to the following principles: (I) The fluorescence yield of o-aminobenzoyl beta-glycerophosphate is increased by 2.75-fold in the presence of saturating concentrations of bovine serum albumin. (II) o-Aminobenzoyl beta-glycerophosphate has the same fluorescence yield as o-aminobenzoyl glycerol, but the latter does not bind to bovine serum albumin. (III) Dephosphorylation of the substrate, catalyzed by the monophosphatase, makes less o-aminobenzoyl beta-glycerophosphate available for binding to bovine serum albumin, thereby producing a decrease in the fluorescence intensity.     

Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

The participation of the striatum in the central regulation of gonadal hormonal function]

Intrastriatal corticotrophin-releasing hormone (CRH) was shown to induce a decrease in the plasma tectosterone concentration. Besides, the 6-OHDA pre-treatment completely prevented the suppression of plasma testosterone in response to intrastriatal CRH administration. The findings suggest that the striatum is involved in the extrahypothalamic regulation of the gonadal endocrine function.     

Evaluation of the efficiency of measures for sulphidic mine waste mitigation

To control the environmentally detrimental impact of acid rock drainage, two different countermeasures, layers of acid-buffering materials and sodium dodecyl sulphate addition, were tested for their efficiency in laboratory percolation experiments. In the experiment with a layer of calcium bentonite, only the iron output was reduced. The experiments with layers of concrete grains demonstrated a decrease of the microbial activity as well as a precipitation of heavy-metal ions, whereas the cell numbers did not decrease. Furthermore, finely grained concrete (1–5 mm) formed a water-tight hardpan (self-sealing layer). In the experiment with 1 mM sodium dodecyl sulphate, all the microorganisms were killed and hence metal sulphide dissolution was stopped. With 0.1 mM sodium dodecyl sulphate only a short, transient inhibition of leaching was achieved. The bacteria remained alive. Received: 16 February 1998 / Accepted: 23 February 1998     

Cathepsin B from human renal cortex.

Cysteine-proteinase activity was observed in homogenates of human-cadaver renal cortex. This activity co-purified with renin enzymic activity until separation by aminohexyl-Sepharose--pepstatin affinity chromatography. The cysteine proteinase was purified 1780-fold after the following successive chromatographic procedures: Sephadex G-75, DEAE-cellulose DE-52, and an organomercurial affinity resin. The proteinase activity was dependent upon activation by thiol-containing compounds such as dithiothreitol, as well as by EDTA, and was inhibited by the thiol-group-specific alkylating reagents iodoacetic acid and N-ethylmaleimide. DE-52 cellulose chromatography resolved the cysteine proteinase into two components. On the basis of molecular size (26 000 daltons), activity as a function of pH, stability as a function of pH, substrate specificity and thermal lability, the major component (95%) has been identified as cathepsin B. The DE-52 cellulose elution pattern of the minor component (5%) is suggestive of cathepsin H [Schwartz & Barrett (1980) Biochem. J. 191, 487-497] Enzymic activity was determined with synthetic substrates, in particular alpha-N-benzoyl-DL-arginine 2-naphthylamide (Bz-Arg-NNap), thus precluding the detection of cathepsin L [Kirschke, Langner, Wiederanders, Ansorge, Bohley & Broghammer (1976) Acta Biol. Med. Germ. 35, 285-299]. Inhibition by dimethyl sulphoxide was observed in the determination of Km = 7.0 +/- 0.4 mM for the substrate Bz-Arg-NNap, and care must therefore be taken in the preparation of substrate solutions.