The role of carboxyl groups on the chitosanase and CMCase activity of a bifunctional enzyme purified from a commercial cellulase with EDC modification

The carboxyl groups of the bifunctional cellulase–chitosanase (CCBE), purified from a commercial cellulase prepared from Trichoderma viride were modified using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC). The EDC modified CCBE lost 80–90% of its chitosnase activity and 20% of its carboxylmethyl cellulase (CMCase) activity; meanwhile, its conformation changed slightly, which altered the substrate binding affinity to chitosan, without affecting its binding to CMC. However, the modification did not alter the structure integrity. The dynamic analysis of modification indicated that the CCBE possessed two carboxylates essential for its chitosanase activity and one carboxyl group for its CMCase activity. One of the two carboxylates involved in chitosanase activity was deduced to be the proton donator, and the other may function for substrate recognition, while the only catalytic carboxyl group for CMCase activity probably also acted as a proton donator.     

Intramolecular modification of azisobutyrylinsulin and the use of the modified hormone for the synthesis of polymeric derivatives

An intramolecular modification of insulin at the alpha-amino group of glycine (A1) and the epsilon-amino group of lysine (B29) was carried out. The modification resulted in a slight alteration of the insulin secondary structure; the modified hormone possessed a biological activity which was practically identical to that of the natural hormone. Therefore the modified insulin can be used as a high molecular weight physiologically active radical inducer for the synthesis of (A1-B29) polyvinylimidazole derivatives. The molecular weight of the covalently linked polymer can be variable. It was shown that the increase in the amount of modifying polymer in the conjugate results in stabilization of the insulin secondary structure concomitant with a decrease of the biological activity and, moreover, of the immunoresponsiveness of the hormone.     

Structural studies on the diglyceride-mediated activation of protein kinase C

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.     

Regulation of the oxidative NADP-enzyme tissue levels in Drosophila melanogaster. I. Modulation by dietary carbohydrate and lipid.

Wild-type third instar larvae of Drosophilia melanogaster fed a casein-sucrose synthetic diet supplemented with phosphatidylcholine (4 mg/ml) possessed 33% more tissue lipid and a modified fatty acid profile compared to larvae fed a fat free-sucrose diet. The rates of lipid synthesis and pentose shunt activity were 2.1 and 2.2 times greater respectively in larvae fed the fat free-sucrose diet than in fat-sucrose fed animals. The tissue concentrations of acetyl-CoA and acyl-CoA were 80 and 61% higher respectively, CoA 49% lower, and the NADPH/NADP+ ratio greater in fat-sucrose fed larvae than in larvae fed a fat free-sucrose diet. Thus, larvae effectively utilized dietary lipid for lipid synthesis and as a supplementary energy source to carbohydrate.     

Chemically modified symmetric and asymmetric duplex RNAs: an enhanced stability to nuclease degradation and gene silencing effect

The present study accents on the relationship between dicing, nuclease stability, and RNAi activity of various types of chemically modified symmetric and asymmetric dsRNAs, covalently bound with amino-groups or cholesterol at one or both terminals. All modified dsRNAs were subjected to cleavage by recombinant Dicer enzyme. They possessed a high resistance to nuclease degradation in cell cultured medium and an excellent RNAi activity in viable cells. The best stability and RNAi activity was detected for 5′-sense amino-modified RNAs. These modifications manifested also a high long-term gene silencing effect within seven days post-transfection, while the RNAi activity of the native 21nt siRNA expired within two days. The conjugation of dsRNA with cholesterol at 5′-sense end resulted in easy intracellular delivery without transfection reagents. After a direct transfection in cells, the cholesterol-conjugated 27nt dsRNA possessed a higher RNAi activity than cholesterol-conjugated 21nt siRNA.     

Comparison of different methods of glucose oxidase immobilization

Glucose oxidase was immobilized by covalent bond to two basic types of sorbents—glycidylmethacrylate copolymers and bead cellulose. These two types of carries were chemically modified, if needed, by the employing various procedures and subsequently used in the immobilization of native and oxidized glucose oxidase. The samples thus obtained were compared with those of immobilized glucose oxidase bound onto some common carriers. Samples which possessed not only a high absolute activity but also adequate mechanical and flow properties were characterized in greater detail with respect to the immobilization efficiency and kinetic properties of bound glucose oxidase.     

Asparagine metabolism and asparaginase activity in a euryhaline Chlamydomonas species.

A Chlamydomonas species isolated from a marine environment possesses an L-asparaginase, an enzyme not yet reported in the microalgae. This enzyme enabled the organism to grow as well with asparagine as sole nitrogen source as with inorganic nitrogen sources (NO3-, NH4+). Only the amide nitrogen was used for growth since growth did not occur on aspartate and aspartate accumulated in the media when cells were either grown on asparagine or during short-term incubations with L-[U-14C]asparagine. Cells grown on NO3-, NH4+, or L-asparagine in batch culture possessed equivalent asparaginase activities. However, nitrogen-limited cells possessed four times the activity of cells grown with sufficient nitrogen for normal growth, regardless of the possessed the lowest activity per cell, while lag phase and stationary phase cells possessed greater activity. The enzyme behaved like a periplasmic space enzyme since (1) breaking the cells did not release into solution more activity than was shown by whole cells and (2) whole cells converted L-[U-14C]asparagine to [14C]aspartate with little intracellular accumulation of radioactivity. Cell-free preparations of the enzyme possessed a Km value for asparagine of 1.1 x 10-4 M, with no glutaminase activity.     

Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes

In adipocytes, amino acids stimulate the target of rapamycin (TOR) signaling pathway leading to phosphorylation of the translational repressor, eIF-4E binding protein-I (4E-BP1), and ribosomal protein S6. L-leucine is the primary mediator of these effects. The structure-activity relationships of a putative L-leucine recognition site in adipocytes (LeuR(A)) that regulates TOR activity were analyzed by examining the effects of leucine analogues on the rapamycin-sensitive phosphorylation of the translational repressor, eIF-4E binding protein-I (4E-BP1), an index of TOR activity. Several amino acids that are structurally related to leucine strongly stimulated 4E-BP1 phosphorylation at concentrations greater than the EC(50) value for leucine. The order of potency was leucine > norleucine > threo-L-beta-hydroxyleucine approximately Ile > Met approximately Val. Other structural analogues of leucine, such as H-alpha-methyl-D/L-leucine, S-(-)-2-amino-4-pentenoic acid, and 3-amino-4-methylpentanoic acid, possessed only weak agonist activity. However, other leucine-related compounds that are known agonists, antagonists, or ligands of other leucine binding/recognition sites did not affect 4E-BP1 phosphorylation. We conclude from the data that small lipophilic modifications of the leucine R group and alpha-hydrogen may be tolerated for agonist activity; however, leucine analogues with a modified amino group, a modified carboxylic group, charged R groups, or bulkier aliphatic R groups do not seem to possess significant agonist activity. Furthermore, the leucine recognition site that regulates TOR signaling in adipocytes appears to be different from the following: (1) a leucine receptor that regulates macroautophagy in liver, (2) a leucine recognition site that regulates TOR signaling in H4IIE hepatocytes, (3) leucyl tRNA or leucyl tRNA synthetase, (4) the gabapentin-sensitive leucine transaminase, or (5) the system L-amino acid transporter.     

Action of different types of gramicidin S derivatives on bacterial cells and protoplasts

The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts. The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells. However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis. Hence, these compounds preserved a certain membranotropic level. Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts.     

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.     

Trichinella spiralis: Generation in the presence of rat serum of factors chemotactic for rat cells

Chemotaxis of rat peritoneal cells, of which the eosinophil was the predominant migratory cell type, toward incubates of Trichinella spiralis was studied using a modified Boyden chamber. Excysted muscle larvae, preadults, and adults were incubated in a buffered medium for 20 hr at 37 C. Worms were incubated alone or with serum or spleen cells, or both, from immune and nonimmune rats. Incubates of worm stages alone possessed no chemotactic activity as compared with incubation medium as a negative control and zymosan-activated serum as a positive control. Both normal and immune sera tested alone stimulated cell migration to the same degree. Incubates of spleen cells from either normal or immunized hosts did not show chemotactic activity. Chemotaxis caused by normal and immune sera were not altered by incubation with homologous spleen cells. Addition of larva, preadults, and adult worms to sera, however, enhanced chemotactic activity over sera alone. Chemotaxis caused by larvae plus immune sera was significantly greater than that stimulated by larvae plus normal sera. This difference decreased when preadults were substituted for larvae and was not observed when adult worms were used. Reversal of the chemical gradients showed that active cell migration caused by various incubates was due to Chemotaxis.     

Virion-Bound Protein Kinase in Semliki Forest and Sindbis Viruses

Semliki forest virus and Sindbis virus (Alphaviruses belonging to the togavirus group) grown in BHK-21 cells possessed very low levels of virion-associated protein kinase activity. For comparison, vesicular stomatitis virus, also grown in BHK-21 cells, contained a virion-bound protein kinase which had a specific activity 80 times greater than that of the Alphaviruses. The Alphavirus protein kinase was unmasked by the nonionic detergent Nonidet P-40 but was not activated by cyclic nucleotides. Phosvitin was the best exogenous phosphate acceptor for assaying the viral enzyme in vitro. Phosphoprotein phosphatase activity was also detected in the Alphaviruses. Both in vivo and in vitro, all of the viral structural polypeptides were phosphorylated, and the phosphorylated amino acids were found to be serine and threonine. The viral nucleocapsid protein was about four times more efficient as a phosphate acceptor than were the envelope proteins. From 33 to 50% of the total protein kinase was bound to the viral nucleocapsid, and the specific activity of this enzyme was 4 to 10 times greater than that associated with the viral envelope.     

Beta-adrenergic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0--8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the beta-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na+K+(Mg2+)-ATPase activity and a 6--8 fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2--3 fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological beta 2 type response with isoproterenol (1.8 . 10(-8) M) > epinephrine (1.1 . 10(-7) M) > norinephrine (3.2 . 10(-6) M). In contrast, binding studies employing (-)-[3H]dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (-)-[3H]dihydroalprenolol by catecholamine agonists. Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of Mr 80000 and 86000, whereas in the adult only a single Mr 113000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.     

Alteration of the ecto-protein phosphorylation profile of intact goat spermatozoa during epididymal maturation

Intact cauda-epididymal mature and caput-epididymal immature goat spermatozoa were assessed for their capacity to phosphorylate the outer surface proteins upon incubation in a modified Ringer's solution containing [delta-32P]ATP. The immature spermatozoa possessed markedly greater (approximately 7-fold) efficacy to phosphorylate the ecto-proteins than the mature cells. Autoradiographic analysis of the 32P-labelled proteins resolved by SDS-PAGE, showed that multiple sperm ecto-proteins are phosphorylated by an endogenous ecto-cyclic AMP-independent protein kinase (CIK) and the phosphorylation profile of these proteins underwent marked alteration during the epididymal sperm maturation. The intact caput-sperm as well showed nearly 4-fold higher specific activity of ecto-CIK than the cauda-sperm when the kinase activity was estimated using phosvitin as the exogenous protein substrate. The data suggest that the ecto-CIK and its specific protein substrates located on the sperm outer-surface, may have important roles in regulating the epididymal maturation of the male gametes.     

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.     

Effect of prevention of lung inflation on metamorphosis and respiration in the developing bullfrog tadpole, Rana catesbeiana

We tested the hypothesis that respiratory development would be retarded in tadpoles reared in aquaria in which a barrier prevented access to the air-water interface. To test this hypothesis, we examined swimming behavior and respiration in intact tadpoles and gill and lung respiratory activity and central chemosensory responses in an in vitro brainstem preparation. The "barrier" tadpoles had significantly lower resting gill frequencies and higher lung breath attempts than control tadpoles at the same metamorphic stage. Control tadpoles swam greater distances and spent more time in the upper one third of the aquaria, while barrier tadpoles spent significantly more time at the bottom of the aquaria. There was significantly greater mortality for barrier tadpoles compared to control animals in the earliest and latest metamorphic stages. Mean body weight was significantly greater, and metamorphic rate was reduced in barrier tadpoles. Neither control nor barrier tadpole brainstem preparations demonstrated a gill ventilatory response to CO(2); however, both control and barrier preparations possessed significant lung frequency responses to central CO(2) chemoreceptor stimulation. Bath application of the GABA(A) and glycine receptor antagonists, bicuculline and strychnine, had greater effects on control tadpole gill burst activity and produced a similar large-amplitude bursting pattern in both control and barrier tadpoles, that was insensitive to CO(2) chemoreceptor stimulation. We conclude that development of the respiratory pattern was perturbed by the barrier, but the major effect was on gill ventilation rather than lung ventilation as we had expected.     

Fermentation of pectin and glucose,and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.     

Utilisation of Glucose by Mycoplasma Suipneumoniae and Mycoplasma Flocculare

The utilisation of glucose by Mycoplasma suipneumoniae and Mycoplasma flocculare was examined by chemical determination of glucose disappearance during growth, and by examination for hexokinase activity in cell preparations. Both species degraded glucose during growth and possessed hexokinase activity as evidence of the presence of a glycolytic pathway. The glucose utilisation capacity was found to be greater for M. flocculare than for M. suipneumoniae.     

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

化学修饰解除天冬酰胺酶抗原性研究

本文报道了用活化的单甲氧基聚乙二醇PEG_2在底物保护的条件下修饰天冬酰胺酶。结果,修饰酶在抗原抗体结合能力完全消失的同时,酶活力保持30％以上,且修饰酶的抗胰蛋白酶水解能力明显增强,体外半衰期延长17倍,免疫原性显著下降。