Inactivation of S-adenosylhomocysteine hydrolase with haloethyl and dihalocyclopropyl esters derived from homoadenosine-6'-carboxylic acid

In a search for new inhibitors that exploit 5'-6' 'hydrolytic activity' of AdoHcy hydrolase, a new series of haloethyl and dihalocyclopropyl esters 2-3 were designed and their interaction with the enzyme studied. Incubation of the enzyme with 2-3 resulted in time- and concentration-dependent inactivation of AdoHcy hydrolase as well as almost total depletion of its NAD(+) content. Further results indicated that the 'oxidative' but not the 'hydrolytic' activity was involved in the inactivation process.     

Evolution of amino acid biosynthesis and enzymes with broad substrate specificity.

I selected 82 proteins that were related to amino acid biosynthesis in the genome of Escherichia coli. I then searched the extensive sequence homology for each of the selected proteins from among the proteins of E.coli. The result showed that 30 proteins of the selected proteins had extensive sequence homology within the selected proteins, and 21 proteins had extensive sequence homology to proteins outside the selected proteins. In addition, the enzymes with broad substrate specificity play an important role in the amino acid biosynthesis. I demonstrate here that some substrate-specific enzymes evolved from an ancestor enzyme with broad substrate specificity. CONTACT: hnishida@iam.u-tokyo.ac.jp     

酵母天然酶系生物合成谷胱甘肽

利用酵母细胞本身具有的谷胱甘肽（GSH）合成酶和ATP合成GSH。在含有半胱氨酸、谷氨酸、甘氨酸的反应液中,加入已充分洗涤的酵母细胞,37℃孵育8h后,酵母体内可积累15mg/g(dry cell weight)以上的GSH,而对照组仅5mg/g(dry cell weight)。同时也研究了树脂法和铜盐法相结合分离纯化GSH。实验结果初步表明,利用酵母细胞中的天然谷胱甘肽合成酶和ATP,可使前体物质快速转化成GSH,是可行的。     

Role of cyclic adenosine monophosphate in the induction of endothelial barrier properties

Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endothelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration-dependent 2-6-fold increase in cyclic AMP which was associated with a 3-10-fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier-enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)-dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp-cAMPS, cyclic AMP-dependent protein kinase agonists. Furthermore, the forskolin effect on barrier enhancement was partially reversed with Rp-cAMPS, an antagonist of cyclic AMP-dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP-induced effects were associated with increases in the polymerized actin pool (F-actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F-actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F-actin, suggesting that influences on cytoskeletal assembly may be involved in this process.     

Amino acid racemase with broad substrate specificity,its properties and use in phenylalanine racemization

Summary Broad substrate specificity amino acid racemase (EC 5.1.1.10) was purified from a crude extract of Pseudomonas putida SCRC-744 to near homogeneity. The enzyme has an isoelectric point of 7.6 and a molecular weight of 62,000–65,000. The enzyme showed a broad substrate specificity toward amino acids, utilizing d-glutamine as the best substrate. d-Phenylalanine acted as a substrate to 1% the velocity for d-glutamine. Maximal reaction velocities were observed at 50°–60°C and around pH 8. The apparent K_m values for d-glutamine and d-phenylalanine were 7.8 mM and 25.7 mM, respectively. Both enantiomers of phenylalanine were efficiently racemized by acetone-dried cells of P. putida SCRC-744.     

Evidence against the presence of 3',5'-cyclic adenosine monophosphate and relevant enzymes in Lactobacillus plantarum

Analysis of cells of Lactobacillus plantarum, starved or undergoing induction, showed no 3', 5'-cyclic adenosine monophosphate (cAMP). Neither adenyl cyclase nor 3', 5'-cAMP phosphodiesterase was detected in extracts. Extracts of L. plantarum did not inhibit these two enzymes of Escherichia coli K-12, strain W1435. Incubation of adenosine triphosphate (ATP)-U-(14)C with cells or various cell-free fractions of L. plantarum did not produce labeled 3', 5'-cAMP. Of various 3', 5'-cyclic and acyclic nucleotides tested, only 3', 5'-cAMP, ATP, and yeast adenylic acid stimulated l-arabinose isomerase. Yeast adenylic acid was two to four times as effective as 3', 5'-cAMP or ATP. 2', 3'-cAMP was not effective.     

A microassay for the peptidase activity of enzymes utilizing ribonuclease S peptide as a substrate

A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced.     

Effect of adenosine protonation on its interaction with polyuridylic acid

The effect of adenosine protonation on complex formation between poly(U) and adenosine has been studied by UV spectroscopy, titration and equilibrium dialysis techniques. A method has been developed to estimate the "misincorporation" of ionized monomer molecules into a polynucleotide--monomer complex. The method is based on combining the titration and binding data. Using this method it is shown that protonated adenosine interacts to some extent with poly(U) in the course of A.2 poly(U) dissociation at acidic pH. Qualitative differences between the effects of ionization of the polymer and monomer components on polynucleotide--monomer interaction are discussed.