The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Effects of exercise on insulin binding and glucose metabolism in muscle

To elucidate the mechanism of enhanced insulin sensitivity by muscle after exercise, we studied insulin binding, 2-deoxy-D-[1-14C]glucose (2-DOG) uptake and [5-3H]glucose utilization in glycolysis and glycogenesis in soleus and extensor digitorum longus (EDL) muscles of mice after 60 min of treadmill exercise. In the soleus, glycogenesis was increased after exercise (P less than 0.05) and remained sensitive to the action of insulin. Postexercise insulin-stimulated glycolysis was also increased in the soleus (P less than 0.05). In the EDL, glycogenesis was increased after exercise (P less than 0.05). However, this was already maximal in the absence of insulin and was not further stimulated by insulin (0.1-4 nM). The disposal of glucose occurred primarily via the glycolytic pathway (greater than 60%) in the soleus and EDL at rest and after exercise. The uptake of 2-DOG uptake was not altered in the soleus after exercise (4 h incubation at 18 degrees C). However, with 1-h incubations at 37 degrees C, a marked increase in 2-DOG uptake after exercise was observed in the soleus (P less than 0.05) in the absence (0 nM) and presence of insulin (0.2-4 nM) (P less than 0.05). A similar postexercise increase in 2-DOG uptake occurred in EDL. Despite the marked increase in glucose uptake and metabolism, no changes in insulin binding were apparent in either EDL or soleus at 37 degrees C or 18 degrees C. This study shows that the postexercise increase of glucose disposal does not appear to be directly attributable to increments in insulin binding to slow-twitch and fast-twitch muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.     

甲型肝炎病毒对理化因子的稳定性

用放射免疫成斑法研究了甲型肝炎病毒FM-175株的理化稳定性和灭活条件,证明甲型肝炎病毒理化稳定性与其它肠道病毒相似,具有广范围的pH(2～10)稳定性;Mg~(++)和Ca可增强其热稳定性,不能抵抗冷冻干燥,但对热的抵抗力明显高于普通肠道病毒,可被紫外线迅速杀灭,也可被多种消毒剂如3～8％的甲醛液,50～90％的乙醇,2％的石炭酸及400ppm的有效氯等杀灭,但可抵抗0.1％甲醛液,2～5％的来苏儿及200ppm的有效氯1小时以上,本研究工作为甲型肝炎病毒的理化性状、保存及消毒条件等提供了实验数据。     

应用酶联免疫吸附试验检测马铃薯卷叶病毒

以辣根过氧化物酶标记马铃薯卷叶病毒抗体,采用双抗体夹心ELISA方法鉴定了马铃薯和洋酸浆的茎、叶、根及马铃薯块茎中的马铃薯卷叶病毒(Potato Leafroll Virus,PLRV),结果表明,对提纯的PLRV可测出的最低浓度为25ng/ml,当包被抗体浓度为40μg/ml、酶标记抗体稀释度为1/120时,可测出马铃薯茎、叶和根汁液中的PLRV,感染PLRV的洋酸浆茎、叶和根汁液的消光值,均比无病对照者高二倍以上,虽然感染PLRV的马铃薯休眠块茎维管束组织汁液的消光值高于无病毒对照,且脐部维管束组织消光值高于顶端,但测定打破休眠的感病块茎顶端维管束组织的阳性结果更为可靠和明显。     

Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the closed macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.