Exocellular mucopolysaccharide closely related to bacterial floc formation.

A bacterium isolated from activated sludge formed a visible floc and also produced an exoenzyme that could bring about deflocculation. Scanning electron microscopic examination revealed that the cells were embedded in a film mesh in the floc, which disappeared after treatment with the deflocculating enzyme. Polysaccharides isolated from the floc were fractionated into three fractions by diethylaminoethyl-Sephadex A-25 column chromatography, whereas those from the free cells were fractionated into only two fractions. The missing fraction was a mucopolysaccharide composed of glucosamine, glucose, mannose, galactose, and rhamnose and was hydrolyzed to oligosaccharides by the deflocculating enzyme. The other two fractions were resistant to the enzyme. These results show that the mesh structure of the floc is dependent on a mucopolysaccharide hydrolyzed by the deflocculating enzyme.     

Isolation of Deoxyfructosazine and Its 6-Isomer from the Nondialyzable Melanoidin Hydrolyzate

The nondialyzable melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was hydrolyzed. The hydrolyzate was fractionated by DEAE-cellulose column and Dowex 50 W column. Deoxyfructosazine and its 6-isomer were respectively isolated from main two fractions, and identified. Even on boiling the melanoidin in aqueous solution, these pyrazines as well as imidazoles and β-hydroxy pyridines in the melanoidin were liberated.

Furthermore, amounts of these heterocyclic compounds liberated from the nondialyzable melanoidin and the fractionated melanoidins (fractionated into five fractions on DEAE-cellulose column according to the method described previously) were examined.

The results obtained seem to suggest that these heterocyclic compounds are not present probably as a molecular skelton or an inclusion compound in the melanoidin, but as a small moiety of the melanoidin molecule with loose chemical bond.     

A method for the isolation of cross-linked nucleosides from DNA: application to cross-links induced by nitrous acid.

A procedure is reported for the isolation of cross-linked nucleosides from nitrous acid-treated calf thymus DNA. Cross-linked DNA was hydrolyzed enzymatically with deoxyribonuclease I and snake venom phosphodiesterase and fractionated on a DEAE-Sephadex column. After desalting, the fractions were characterized by ultraviolet spectroscopy, anion exchange high pressure liquid chromatography, gel filtration, and two dimensional thin layer chromatography. A cross-linked dinucleotide, and a series of oligonucleotides were isolated. The oligomers, which had resisted digestion by the above enzyme system, were digested to the nucleoside level by a spleen phosphodiesterase-alkaline phosphatase combination. A second cross-linked product was isolated from this mixture. The cross-linked nucleosides were less than 0.17% of the total nucleotides of the DNA. The methods developed here are recommended for the isolation of products from DNA treated with other cross-linking agents.     

LOCALIZATION AND REACTIONS OF A PHEROMONE DEGRADATIVE ENZYME ISOLATED FROM AN INSECT ANTENNA

A fluid isolated from chemosensory sensilla on the antennaeof males and females of the cabbage looper, Trichoplusia ni(Hübner), was fractionated by gel filtration. Enzymaticdegradation of the pheromone was associated with 2 distinctgroups of fractions. The first group contained membrane fragments,vesicles, and membrane-bound enzyme or insoluble enzyme aggregates.The second group of fractions contained solublilized enzyme.Because of the method of isolating the ‘sensillum liquor’by breaking the tips of the hairs, membranes from chemoreceptorcell dendrites should greatly predominate. In general, 3 isomersand the saturated analog of the pheromone were hydrolyzed morerapidly than the pheromone. ^*Mention of a proprietary product in this paper does not constitutean endorsement of this product by the U. S. Department of Agriculture.     

Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.     

Regulation of influenza virus RNA polymerase activity by cellular and viral factors.

An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.     

The activator of cerebroside sulphatase. Lysosomal localization.

1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Ca(2+)-stimulated organic pyrophosphatase--a new plant phosphohydrolase from wheat seedling microsomes

A new kind of phosphohydrolase, an organic pyrophosphatase (OPPase) was found in microsomes of wheat seedlings. The enzyme was isolated from microsomal membranes with 350 mM KI and separated on Red Sepharose CL 6B into two fractions: OPPase I and OPPase II. OPPase II was partially purified and its physicochemical properties were characterized. The enzyme was of low molecular mass (40 kDa), hydrolyzed pyrophosphoric bonds coupled to organic residues and was activated by divalent cations with a distinct preference for Ca2+. Its activity was insensitive to commonly used inhibitors of all known plant ATPases and acid phosphatases. Some properties of the enzyme are similar to those of potato apyrase (EC 3.6.1.5).     

Ribonuclease of cultured mammalian cells

KB cell ribonuclease has been purified 260-fold and the fundamental properties have been studied. Though the enzyme is concentrated in the lysosomal fraction, appreciable quantities are present in the cell sap and nuclear fractions. Comparison of the optimal temperature and pH for activity, and the heat stability of enzyme from these three fractions suggests that only one species of this enzyme exists in these cells. The enzyme behaves as an endonuclease, cleaving synthetic pyrimidine polynucleotides to smaller oligonucleotides with cyclic 2′:3′ end-groups. The final product is pyrimidine nucleoside 3′ monophosphate. Polyadenylic acid is not hydrolyzed. Of the properties examined in this study only two differences were noted between KB cell and pancreatic ribonuclease. KB cell enzyme acts optimally at pH 6 as opposed to an optimum at pH 7 to 8 for pancreatic enzyme. In addition ribonuclease from KB cells is definitely less stable to heating at 100°C than is the enzyme isolated from pancreas.     

Pyrimidine reducing enzymes of rat liver.

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4. Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH- dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.     

Sub-cellular distribution and isotypes of a 49-kDa apyrase from Pisum sativum

We isolated a 49-kDa protein from various sub-cellular fractions from pea (Pisum sativum L. var. Alaska) stems using heparin affinity and cation exchange column chromatography. The corresponding proteins from all these fractions were identified as apyrase (EC 3.6.1.5) because they hydrolyzed both nucleoside tri- and diphosphates into their respective monophosphates. Using an antibody raised against apyrase, we studied the enzyme’s sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50%), the supernatant (33%), the cytoskeleton (14%), and the nuclei (3%). Immuno-electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures in the cytoplasm associated with ribosomes. Even though there is only one gene (with two alleles), for this protein, 2D gels indicated there were at least five isotypes, three being major, and the relative abundance of these isotypes differed in different fractions. Enzymes from all fractions: (a) hydrolyzed nucleoside triphosphates and diphosphates, but not monophosphates, (b) were insensitive to most ATPase inhibitors (azide, fluoride, nitrate, molybdate, ouabain, quercetin), but (c) were all inhibited by vanadium pentoxide at relatively high concentrations. There were, however, some subtle differences between enzymes from different sub-cellular fractions, including different ADP/ATP hydrolysis ratios. These results show that the 49-kDa apyrase is located in various compartments within the cell (cell wall, nuclei, and the cytoskeleton) and that the enzymes from all fractions are basically similar in their apyrase function. We suggest that the enzyme is modified in various ways to furnish different forms with different (non-apyrase) functions in different sub-cellular locations.     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

Studies on the competence-inducing factor of Bacillus subtilis

1. Aqueous extracts of competent cells of Bacillus subtilis 168I(-) were shown to contain a competence-inducing factor. The aqueous extracts were fractionated on DEAE-cellulose columns. 2. Those fractions from DEAE-cellulose columns containing the competence-inducing factor were shown to exhibit a powerful lytic effect on isolated cell walls of B. subtilis 168I(-). Little or no lytic activity was exhibited by the other fractions. 3. The kinetics of the lytic enzyme were investigated and found to be first-order. Treatment of cell wall lysates with 1-fluoro-2,4-dinitrobenzene suggested that the enzyme may be identical with N-acetylmuramyl-l-alanine amidase. The amino acid composition of the partially purified enzyme was determined. 4. It is suggested that competence-induction may be dependent on the limited action of the autolytic amidase.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells

Heparan sulfate was isolated form the cell surface, cell pellet, and culture medium of exponentially growing as well as postconfluent bovine aortic smooth muscle cells (SMCs). After chromatography on DEAE-Sephadex and Sepharose 4B, the various mucopolysaccharides were examined for their ability to cause growth inhibition in a SMC bioassay. The heparan sulfate isolated from the surface of postconfluent SMCs possessed approximately eight times the antiproliferative potency per cell of the heparan sulfate obtained from the surface of exponentially growing SMCs. Heparan sulfate isolated from other fractions of exponentially growing or postconfluent SMCs possesses little growth inhibitory activity. The difference in the antiproliferative activities of heparan sulfate obtained from the surface of SMCs in the two growth states could not be attributed to the synthesis of a greater mass of mucopolysaccharide by postconfluent SMCs. Indeed, heparan sulfate isolated from the surface of the postconfluent SMCs exhibits a specific antiproliferative activity which is 13-fold greater than mucopolysaccharide obtained from the surface of exponentially growing SMCs and more than 40-fold greater than commercially available heparin. In addition, exponentially growing SMCs did not exhibit an enhanced ability to degrade the complex carbohydrate. Furthermore, other investigations indicate that the small amount of growth inhibitory activity intrinsic to heparan sulfate isolated from the surface of exponentially growing SMCs is due to residual, biologically active, mucopolysaccharide produced by the primary postconfluent SMCs from which the exponentially growing SMCs were derived. These studies suggest that bovine aortic SMCs are capable of controlling their own growth by the synthesis of a specific form of heparan sulfate with antiproliferative potency.     

Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074.

During the course of the transformation of 1,3-dichloro-2-propanol (DCP) into (R)-3-chloro-1,2-propanediol [(R)-MCP] with the cell extract of Corynebacterium sp. strain N-1074, epichlorohydrin (ECH) was transiently formed. The cell extract was fractionated into two DCP-dechlorinating activities (fractions Ia and Ib) and two ECH-hydrolyzing activities (fractions IIa and IIb) by TSKgel DEAE-5PW column chromatography. Fractions Ia and Ib catalyzed the interconversion of DCP to ECH, and fractions IIa and IIb catalyzed the transformation of ECH into MCP. Fractions Ia and IIa showed only low enantioselectivity for each reaction, whereas fractions Ib and IIb exhibited considerable enantioselectivity, yielding R-rich ECH and MCP, respectively. Enzymes Ia and Ib were isolated from fractions Ia and Ib, respectively. Enzyme Ia had a molecular mass of about 108 kDa and consisted of four subunits identical in molecular mass (about 28 kDa). Enzyme Ib was a protein of 115 kDa, composed of two different polypeptides (about 35 and 32 kDa). The specific activity of enzyme Ib for DCP was about 30-fold higher than that of enzyme Ia. Both enzymes catalyzed the transformation of several halohydrins into the corresponding epoxides with liberation of halides and its reverse reaction. Their substrate specificities and immunological properties differed from each other. Enzyme Ia seemed to be halohydrin hydrogen-halide-lyase which was already purified from Escherichia coli carrying a gene from Corynebacterium sp. strain N-1074.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.