Partial purification and properties of a murine plasmacytoma glucosyltransferase

The enzyme UDP-glucose:dolichylphosphate glucosyltransferase has been purified 1700-fold from MOPC-315 plasmacytoma tissue. The purification combines differential detergent extraction of purified rough endoplasmic reticulum with subsequent ion exchange chromatography, dye affinity chromatography, and hydroxylapatite chromatography of the extract. The partially purified glucosyltransferase exhibits a Km of 0.79 microM for UDP-Glc and a Km of 0.65 microM for dolicholphosphate in the presence of 4 mg/ml of phosphatidylcholine. The reaction is dependent upon the addition of exogenous dolicholphosphate. The enzyme is activated by the choline containing lipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The dye Remazol blue acts as a competitive inhibitor of the enzyme with respect to UDP-Glc. The molecular weight of the enzyme has been determined to be approximately 37,000. The sole reaction product has been identified as dolichylphosphate glucose by isolation of the product by DEAE-cellulose chromatography and subsequent analysis of the acid-hydrolyzed product by both Bio-Gel P2 gel filtration and paper chromatography.     

Identification of Ascorbate as an Endogenous Substance That Irreversibly Inhibits Binding of Dihydropyridine Calcium Channel Blockers

Endogenous material present in heat-denatured extracts of rat brain that inhibited the binding of [3H]-isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-metho xyca rbonyl-2,6-dimethyl-3-pyridinecarboxylate ([3H]-PN200-110) to calcium channels in brain membranes was purified. Spectrophotometric analysis of material purified by strong anion-exchange and reverse-phase chromatography showed an absorption maximum at 266 nm at pH 7.0 that shifted to 245 nm at pH 2.0. This pH-dependent spectral shift was indistinguishable from that of ascorbic acid. Samples of the purified extract contained ascorbic acid; however, the inhibition of binding by purified material was always greater than the inhibition seen with equivalent concentrations of ascorbate, implying the presence of additional inhibitory factors. Attempts to detect and identify such inhibitory substances by chromatography showed that inhibition activity was coincident with the presence of ascorbate, and the inhibitory activity of purified material was abolished after treatment with ascorbic acid oxidase. Iron enhanced the inhibition produced by ascorbate, and chemical analysis of purified preparations revealed the presence of iron. Studies comparing the potency of the purified material with that of a mixture of ascorbate plus iron showed that the content of ascorbate and iron in the purified brain extract is sufficient to explain the observed inhibition of binding of [3H]PN200-110.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Purification and properties of monomeric cytochrome f from charlock, Sinapis arvensis L

Monomeric cytochrome f has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, isoelectric focusing and analytical ultracentrifugation, from the leaves of charlock, Sinapis arvensis L. The cytochrome was obtained in an aqueous extract following extraction of leaf lipids with butan-2-one, and was subsequently purified by acetone precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The purified cytochrome had adsorbance ratios of A422/A280 = 7.3 and A554/A280 = 1.07 in the reduced form. There was no indication of the presence of an absorbance band at 695 nm in the oxidised form. The cytochrome had a midpoint redox potential of +365 mV and was oxidised very rapidly by parsley plastocyanin. The molecular weight of the cytochrome was approximately 27 000 as determined by sedimentation equilibrium and by polyacrylamide gel electrophoresis in the presence of dodecylsulphate. The sedimentation coefficient (so20,w) of cytochrome f was 2.48 S. The cytochrome had an isoelectric point at pH 5.50 determined by isoelectric focusing in polyacrylamide gels.     

Preparative method of isolating phosphoinositide fractions from brain tissue]

A simple technique for preparative isolation of chromatographically homogenous fractions of mono-, di- and triphosphoinositides from ox brain tissue is described. Podyphosphoinositides fractions were obtained after chromatography of lipid extract on DEAE cellulose, phosphomonoinositides fraction--after chromatography of polyphosphoinositide-free material on aluminium hydroxide column. Bivalent metal ions were eliminated from lipid extract using chromatography on Dowex-50 H+. Ammonium acetate was removed after precipitation of lipids in water: methanol (1:1) in the presence of 4 M this salt. The average yield of mono-, di- and triphosphates was 40, 22 and 58 mg respectively per 1 kg of brain tissue as callulated for lipid phosphorus.     

Fatty acid distribution of the normal sciatic nerve lipids in the rat

We isolated and purified the main lipids of the rat sciatic nerve. After methanolysis, fatty acids were isolated and purified by thin layer chromatography, and studied by gas chromatography. C 16 and C 18 fatty acids are the most abundant. Among the long-chain fatty acids, arachidonic acid (20:4) is present in some lipids; highly desaturated fatty acids in C 22 and C 24 are also present. In general, the fatty acids are highly saturated; cholesterol esters and ethanolamine phosphoglyceride fatty acids are highly unsaturated.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.     

Influence of extract of Rosa rugosa roots on lipid levels in serum and liver of rats

The effects of the methanol extract of Rosa rugosa roots on serum and liver lipids were studied in rats. The rats were fed the purified diets with or without the methanol extract at the 1% level for 4 weeks. The concentrations of serum and liver total cholesterol were not significantly affected by the feeding of extract. Feeding of the extract, on the other hand, reduced the liver triacylglycerol content without influencing the serum triacylglycerol level. The effects of the extract on lipids profiles were diminished markedly by dietary cholesterol. The results suggest an existence of component in the extract which may ameliorate the accumulation of triacylglycerol in rat liver.     

Determination of brain gangliosides by determination of ganglioside stearic acid

A new method is described for the determination of brain gangliosides by measuring stearic acid, the chief acid of gangliosides, in an appropriately purified brain extract. The method includes extraction of tissue with chloroform-methanol, extraction of gangliosides from the extract with 0.1 M KCl, evaporation of the aqueous phase, methanolysis, and gas-liquid chromatography of the resultant methyl esters with a double internal standard. The method depends on the simple composition of ganglioside fatty acids (80% stearic acid) and allows determination of less than 0.05 micromole of gangliosides. Interfering lipids are removed from the ganglioside extract by washing with chloroform-methanol-water. The effects of contamination with nonlipid N-acetylneuraminic acid are avoided.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.     

Caroteno-protein complexes in Axinella verrucosa (Porifera: Demospongae)

Carotenoproteins are common to a wide variety of animals, most notably invertebrates although other animal groups have been shown to have these complexes as well. In a previous paper alpha-carotene, renieratene, isorenieratene and an unknown isomer of renieratene were isolated, but at present nothing is known about nature of the carotenoid-protein complexes in A. verrucosa. In this paper we report our results about a carotenoid-protein complex extracted by TRITON X-100 and purified by Sephadex G 200 Fine and DEAE-Sephacel chromatography. This carotenoprotein had a molecular weight of ca. 390,000 dalton (Gel electrophoresis). SDS PAGE plate showed only a single polypeptide chain of ca. 50,000 dalton. The proteic band separated by PAGE plate electrophoresis showed the presence of lipids and glycids when stained respectively by Sudan Black B and by Schiff's reagent. The extract obtained without TRITON X-100 showed the presence of a similar carotenoid-protein complex, but because of the low proteic concentration this result should be confirmed.     

Liver microsomal expoxide hydrase. Solubilization, purification, and characterization.

Epoxide hydrase was solubilized from liver microsomes of phenobarbital-treated rats by treatment with cholate and purified to apparent homogeneity by ammonium sulfate fractionation and column chromatography in the presence of the nonionic detergent Emulgen 911 on DEAE-cellulose and hydroxylapatite. The purified enzyme preparation had a single major band with a molecular weight of 53,000 to 54,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Other studies indicated that in the absence of sodium dodecyl sulfate, purified epoxide hydrase exists as high molecular weight aggregates. The preparation was essentially free of heme and flavin, but still contained small amounts of lipids and Emulgen 911.     

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.     

Purification and characterization of alpha-L-fucosidase from Streptomyces species.

Streptomyces sp. 142, isolated from a soil sample, produced alpha-fucosidase when cultured in the presence of L-fucose. The enzyme was purified 700-fold with an overall recovery of 17% from a cell-free extract by cation exchange chromatography and gel filtration chromatography. The apparent molecular weight of the purified enzyme was 40,000 by gel filtration chromatography. The enzyme had a pH optimum of 6.0 and was stable at pH 4.5-7.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine as the substrate showed that the enzyme specifically hydrolyzed terminal alpha 1-3 and alpha 1-4 fucosidic linkages in the oligosaccharides but did not hydrolyze alpha 1-2 or alpha 1-6 fucosidic linkages or a synthetic substrate, p-nitro-phenyl alpha-L-fucoside. The purified enzyme released L-fucose from a fucosylated glycoprotein, alpha 1-acid glycoprotein. Thus, the substrate specificities of the Streptomyces alpha-fucosidase resembled those of alpha-fucosidases I and III isolated from almond emulsin rather than those of other microbial alpha-fucosidases.     

Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain

The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.     

Isolation of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine from the bull heart

A novel efficient procedure based on silica gel column chromatography has been developed for isolating chromatographically pure diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. For this purpose the lipid extract is treated with aqueous MgCl2, whereas aqueous ammonia is added to the eluent systems. To raise the yield of lipids, a fractional loading of columns with portions of lipid extracts is employed, each loading being followed by partial elution of neutral lipids with chloroform. Optimal phospholipid--silica gel ratio is 1:20 for such a procedure. The presence of cation exchange between the lipid extract and silica gel is confirmed and its influence on the efficacy of phospholipid separation is discussed.     

Chromatographic behaviour of rat-liver monophosphoinositide

1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform–methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform–methanol–water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform–methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide.     

Purification and some properties of skeletal muscle sarcolemma Ca2+-ATPase]

Ca2+-ATPase of skeletal muscle sarcolemma has been isolated and purified. It is prepared from salt extract of sarcolemma by ammonium sulfate fractionation and further purified by gel chromatography on Sepharose 4B. The purity of preparations was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has been shown that Ca2+-ATPase possesses the same mobility as skeletal muscle myosin under gel chromatography on Sepharose 4B and the same mobility as myosin heavy chains in sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Membrane protein binds to rabbit skeletal muscle actin, and this complex dissociates by ATP. Interaction with actin does not change Ca2+- or Mg2+-stimulated ATPase activity. Enzyme has only one pH optimum at 7,0-7,6. Membrane protein is highly specified to calcium--ATPase activity in the presence of Mn2+ is 10% and in the presence of Sr2+, Mg2+ or Co2+ are 3-5% of the activity in the presence of Ca2+. Other nucleoside triphosphate (UTP and ITP) are hydrolyzed at lower rates than is ATP.