Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.

Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half-maximal saturation of photoinsertion into the polypeptide hormone was at 8-12 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 microM enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse-phase high performance liquid chromatography (HPLC) revealed that the N-terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N-terminal region is suggestive of a physiological role for a glucagon-GTP complex in the mechanism of action of this hormone.     

垂体后叶素和加压素对离体心肌的直接作用

本实验采用大鼠离体右心房和右心室肌条模型,观察了垂体后叶素和加压素对右心房和右心室肌的直接作用。结果表明:垂体后叶素对右心房的自主性收缩频率和幅度及右心室肌的收缩幅度均有剂量依赖性抑制作用;加压素对右心房和右心室肌收缩幅度也有剂量依赖性抑制作用,但对右心房自主节律无影响;催产素对右心房的收缩频率和幅度则均无影响。加压素V_1、V_2受体拮抗剂d(CH_2)_5Tyr(Me)AVP和d(CH_2)_5(D-Ile~2,Ile~4,Ala(NH_2)~9)AVP对垂体后叶素的负性变力作用具有不同程度的阻断作用,但对垂体后叶素的负性变时作用无阻断作用。以上结果提示,垂体后叶素的负性变力作用主要是由加压素产生的,加压素对心肌有直接的负性变力作用;垂体后叶素的负性变时作用可能是非加压素和催产素成分的作用结果。     

生态因子对中药甘草质量影响的初步研究

甘草是一种常用中药,由于具有“调和诸药”之功效而成为方剂中最常用的一味药。古有“十方九草”之说,可见对甘草的需求量之大;目前,对甘草的需求不仅仅限于医药界,在食品、轻工、畜牧等行业也有广泛的应用。然而,由于长期滥采、滥挖,只挖不种,使得甘草的蕴藏量急剧下降,有些地区的甘草资源濒临枯竭。例如,东北松嫩平原,原是我国“东草”商品的主产区之一,并大量出口国外,     

Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of³²P-phosphate from [-³²P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12.

The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein. In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions. DNA sequence analysis identified 20 different mutations. In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA. In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction.     

Separation of the nativelike intermediate from unfolded forms during refolding of ribonuclease A

In an effort to determine structural properties of the nativelike intermediate (i.e., IN) which forms during the refolding of RNase A, refolding samples were subjected to rapid HPLC gel filtration which allowed us to separate IN from unfolded forms of RNase. The comparison of these samples, enriched in IN and depleted of unfolded forms, with unseparated control samples at the same stage of refolding allowed certain conclusions to be drawn concerning the properties of IN. First, the results show that the transition from IN to native RNase occurs with only small changes in fluorescence. This means that the major fluorescence changes seen during normal refolding experiments must be associated with changes in proline isomerization of unfolded species and/or with the refolding step itself but not with the IN----N step. Second, the fluorescence assay for isomerization of proline-93 shows that IN exists with proline-93 in a state of isomerization identical with or very similar to native RNase; i.e., proline-93 is cis in IN and not trans as suggested by others. All results are semiquantitatively consistent with our earlier refolding model and not nearly so consistent with alternative models which assume that most or all of the slow-refolding forms of RNase have proline-93 in the incorrect trans state.