Primary structure of the S-peptide formed by digestion of rabbit liver fructose 1,6-biphosphatase with subtilisin.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH₂-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala.     

高效液相层析法纯化人胎盘谷胱甘肽硫转移酶

足月分娩的新鲜胎盘组织制成匀浆后,经高速离心、超速离心,谷胱甘肽(GSH)Sepharose 6B亲合层析,Amicon pM-10膜超过滤及高效液相层析,最终经SDS-PAGE鉴定,结果呈现单一亚基区带,其亚基分子量为25000。 根据我们现有高效液相设备条件,用ODS柱代替RadulovicL等报道的特异阴离子柱,用磷酸盐洗脱液代替含谷胱甘肽、二硫苏糖醇及氯化钾的梯度洗脱液,从人胎盘组织成功地制备了谷胱甘肽硫转移酶(GST)纯酶,全过程在15min内完成,保留时间及主峰面积的重复性均较理想,7次实验结果的变异系数为0.2％,最终纯化578.9倍。本研究为各种形式GST的纯化制备提供了一个新的、重复性好、分辨率高及回收理想的简易方法。     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

The acid-catalyzed decompostion of phenacylcobalamin: evidence for the formation of an enol-Co(III) pi-complex intermediate.

Phenacylcobalamin has been synthesized and characterized by thin-layer chromatography and uv-visible spectroscopy, as well as identification of the cobalt-containing and organic products of its cleavage in acid and base and by aerobic photolysis. The major organic product from all three cleavage reactions is acetophenone and the cobalt-containing product is aquacobalamin (or hydroxocobalamin, its conjugate base). In aqueous acidic solution (pH 0 to 7.3, ionic strength 1.0 M, and 25.0 degrees C), the kinetics of the formation of aquacobalamin are biphasic representing the linear sum of two exponential terms. The pH dependence of the first-order rate constant of both phases shows a first-order dependence on proton concentration but with an inflection point ot pH 3.55 for the faster phase and at pH 4.03 for the slower phase. This behavior is interpreted in terms of the specific acid catalyzed formation of an intermediate from both "base on" and "base off" phenacylcobalamin with different second-order rate constants for each form, followed by an intermediate decompotion step with a similar formal mechanism. The nature of the intermediate is discussed and it is concluded to be a pi-complex between cob(III)alamin and the enol of acetophenone.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

The ultrastructure of the middle cerebral artery and its associated nerve fibers in the squirrel monkey and baboon

This study describes the ultrastructural characteristics of the middle cerebral artery and its related neural elements in the squirrel monkey and baboon. The cytoarchitecture of the M-1 segment as well as that of the smaller extracerebral and intracerebral vessels is comparable in both animals.Smooth muscle elements are occasionally found within the intimai lining. The nerve bundles associated with vessels contain fewer myelinated fibers as the vessel diameter decreases. The cytological relationship between the neural structures and the smooth muscle cells are discussed.