Ovine pancreatic lipase : purification and some properties.

Lipase has been isolated from sheep pancreas. The lipoprotein complex formed in pancreas homogenates by the enzyme and endogenous lipids is split by treatment with acetone. Lipase is further purified by ion-exchange chromatography and gel filtration. The molecular weight and the amino-acid composition of ovine lipase are very similar to that of the porcine and bovine enzymes. As previously found in bovine lipase, no carbohydrate is covalently bound to the polypeptide chain which has a N-terminal residue of lysine. The study of the catalytic properties of ovine pancreatic lipase indicates that the enzyme is fully activated by colipase from various species in the presence of conjugated bile salt micellar solutions.     

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase.

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.     

A photochemical method for rapid and precise determination of carbon monoxide levels in blood.

A simple and inexpensive flash photolysis apparatus for determination of the level of carbon monoxide saturation of blood samples is described. Saturation with CO is determined by observing the change in light transmission at 432 nm produced on photolysis of bound CO with a light flash. The procedure is highly specific for carbon monoxide, requires less than 5 μl of blood (obtainable from a finger prick), and has a resolution better than 0.1% in saturation. In addition the apparatus does not require frequent calibration.     

Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, d-strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2′-iminodipropionic acid) were chromatographeo as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 μg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Littorina littorea, and the strombine/alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mm hydrazine buffer, pH 9.0, 5 mm NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.     

A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma.

A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ in blood plasma. The method involves a simplified and more specific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25-dihydroxyvitamin D₃ with the use of polyethylene glycol. The recovery of 1,25-dihydroxyvitamin D₃ during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 ± 2 pg/ml.     

One-step molybdate method for rapid determination of inorganic phosphate in the presence of protein

A colorimetric method for the determination of γ-carboxyglutamic acid (Gla) is presented. The procedure is based on the following results. (a) Gla is quantitatively converted into a proline derivative by reaction with acetaldehyde. (b) This derivative is spectrophotometrically detected by the secondary amine reagent: nitroprusside and acetaldehyde in alkaline medium. Under the reported conditions, Beer's law is obeyed for concentrations of Gla varying from 0.5 to 5 × 10^?4m. The method has been used to determine the Gla content in urine samples.     

Liquid membrane elctrodes for bile salts

A liquid membrane electrode is described which can measure the activity of biological detergents in solution. The results for two bile salts, sodium taurocholate and sodium taurodeoxycholate, are compared with those for a conventional detergent, sodium dodecyl sulfate. The activity of the bile anion is also measured in a mixed micellar solution (phosphatidylcholine added).     

A simple method for the preparation of polyacrylamide gels containing thioglycoside ligands.

A simple method was developed for the preparation of acrylamide derivatives containing thioglycosides. The synthetic scheme involves the preparation of an O-acetylated 1-thiosugar via a pseudothiourea derivative, the controlled addition of the thiosugar to N,N′-bismethyleneacrylamide to form a monoaddition product in which one of the acrylamide groups remains unchanged, and finally de-O-acetylation. Similar reaction schemes using bisacrylamido derivatives of 1,2-diaminoethane and 1,6-diaminohexane lead to analogous compounds with longer aglycon chain lengths. The thioglycoside derivatives of acrylamide thus prepared could be copolymerized with acrylamide to form polymers or gels containing thioglycosides as ligands. These gels were successfully used as affinity materials for the purification of peanut lectin and in cell-adhesion studies.     

Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells.

Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.     

Studies on the action of nerve growth factor: I. Characterization of a simplified in vitro culture system for dorsal root and sympathetic ganglia

Neurite outgrowth from dorsal root (DRG) and sympathetic ganglia has been studied utilizing a simplified in vitro culture system for intact ganglia. Attachment of ganglia to tissue culture plates was achieved after a brief incubation of ganglia on the plates in the presence of 100% fetal calf serum or 5% ovalbumin in F12 medium. Neurite outgrowth from dorsal root and sympathetic ganglia was dependent on the continued presence of nerve growth factor (NGF) and on the NGF concentration. The NGF induced neurite outgrowth from DRG cultured in serum-free medium was delayed approximately 24 hr compared to the outgrowth in serum-containing medium.     

A simple method for quantitative, semiquantitative, and qualitative assay of protein.

Aqueous cesium trichloroacetate permits the buoyant resolution of various RNAs and also of DNA at room temperature and neutral pH. Precipitate formation does not occur, under either native or denaturing conditions. The compositional buoyant density gradient was determined, and the buoyant densities of a variety of RNAs are presented. The buoyant densities increase in the order protein < DNA ? duplex RNA ? single-stranded RNA.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.     

A new method for the determination of N-sulfate in heparin and its analogs.

A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.