Synthesis of 7-mercaptoheptanoylthreonine phosphate and its activity in the methylcoenzyme M methylreductase system

The structure of component B of the methylcoenzyme M methylreductase of Methanobacterium thermoautotrophicum was recently assigned as 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (Noll, K. M., Rinehart, K. L., Jr., Tanner, R.S., and Wolfe, R.S. (1986) (Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242). We report here the chemical synthesis and biochemical activity of this compound. Thiourea and 7-bromoheptanoic acid were used to to synthesize 7,7'-dithiodiheptanoic acid. This disulfide was then condensed with DL-threonine phosphate using N-hydroxysuccinimide and dicyclohexylcarbodiimide. The product was reduced with dithiothreitol to give HS-HTP. It could be oxidized in air in the presence of 2-mercaptoethanol to give the compound as it was isolated from cell extracts. The resulting product was identical to the authentic compound by 1H NMR spectroscopy, mass spectrometry, and coelution using high performance liquid chromatography. The synthetic compound is active in the in vitro methanogenic assay at concentrations comparable to the authentic compound. This confirms the structure of component B as HS-HTP and provides a means to synthesize quantities sufficient for studies of the methylreductase system.     

Changes in the Activity of the Maturation-Promoting Factor Are Correlated with Those of a Major Cyclic AMP and Calcium-Independent Protein Kinase During the First Mitotic Cell Cycles in the Early Starfish Embryo

Many studies suggest that MPF activation depends on protein phosphorylation or that MPF is itself a protein kinase. In the present report, cyclic variations of MPF activity have been correlated in vivo with changes in the extent of protein phosphorylation or in vitro with changes of a major protein kinase during the first cell cycles of fertilized starfish eggs. This cycling protein kinase neither requires cAMP nor Ca²⁺. Neither colchicine nor aphidicoline, which inhibits cleavage and chromosome replication respectively, was found to suppress the synchronous and cyclic variations of both MPF and protein kinase activities. Protein synthesis was found to be required for both MPF and protein kinase activities to reappear after their simultaneous drop at the time of mitotic or meiotic cleavages. Production of either MPF or protein kinase activities is not the immediate result of protein synthesis since there is a delay at each cell cycle between the time when protein synthesis is required and the time when both MPF and protein kinase activities are produced. This suggests that both MPF and protein kinase activities might involve some post-translational modification of a precursor protein synthesized during the preceeding cell cycle.     

Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.

The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.     

中国平脉树螽属五新种记述：直翅目：螽斯科：树螽亚科

本文报道中国平脉树螽属5个新种。每个新种皆有详细的形态描述和形态特征图。所有模式标本存于北京农业大学昆虫标本室。     

Cellularity of organs in mature rams of different breeds

The relative importance of cell number and cell size in determining the mass of 16 organs and tissues in mature rams of six different breeds was studied through estimation of organ deoxyribonucleic acid (DNA) content. The mean fleece-free empty body weight (FFEBW) ranged from 54.6 +/- 0.3 kg for Camden Park Merinos to 76.7 +/- 1.6 kg for Strong Wool Merinos. For all organs, mass increased with FFEBW, but the relationship was significant across all sheep for only eight organs (blood, kidney, liver, abomasum, vastus lateralis muscle, skin, perirenal fat and triceps muscle). There were significant differences between breeds in the mass of 11 organs. With four (heart, rumen reticulum, small intestine and testicular fat) this difference was independent of breed differences in FFEBW, whereas with another four (kidney, abomasum, vastus lateralis muscle and skin), it was closely related to FFEBW. Breed differences in the mass of the remaining three organs (blood, liver and perirenal fat) were partly related to FFEBW and partly breed specific. Blood mass increased with FFEBW across all animals, but, within a breed, it declined as FFEBW increased. The increase in the mass of perirenal fat with FFEBW was significantly greater within a breed than between breeds. Cell number increased significantly with the mass of all organs except blood and brain. There were between-breed differences in the number of cells in seven organs (liver, heart, rumen reticulum, abomasum, small intestine, vastus lateralis muscle and skin), which, except for heart, were attributable to between-breed differences in organ mass. With heart, the increase in cell number with organ mass within a breed was greater than across all breeds. Cell size was significantly related to organ mass only with vastus lateralis muscle, spleen, perirenal fat and liver. The relationship for vastus lateralis muscle and spleen was negative, indicating that cells were smaller in larger organs. There were differences between breeds in cell size for heart, vastus lateralis and triceps muscles. These differences for heart and triceps muscle were breed specific, whereas for vastus lateralis muscle it was attributed to breed differences in organ weight. There was a 30-fold range in mean cell size across organs, with adipose tissue having the largest cells, muscle tissue intermediate and visceral tissues the smallest. In general, organ mass is positively related to FFEBW. Cell number, not cell size, is largely responsible for differences in organ mass between mature sheep of different breeds.     

Inhibition of cathepsin B by peptidyl aldehydes and ketones: slow-binding behavior of a trifluoromethyl ketone

Inhibition of the cysteine proteinase cathepsin B by a series of N-benzyloxycarbonyl-L-phenylalanyl-L-alanine ketones and the analogous aldehyde has been investigated. Surprisingly, whereas the aldehyde was found to be almost as potent a competitive reversible inhibitor as the natural peptidyl aldehyde, leupeptin, the corresponding trifluoromethyl ketone showed comparatively weak (and slow-binding) reversible inhibition. Evaluation of competitive hydration and hemithioketal formation in a model system led to a structure-activity correlation spanning several orders of magnitude in both cathepsin B inhibition constants (Ki) and model system equilibrium data (KRSH,apparent).