Unusual solvent effect on protease activity and effective optical resolution of amino acids by hydrolytic reactions in organic solvents

Summary The catalytic activities of -chymotrypsin, subtilisin Carlsberg, and subtilisin BPN' for hydrolysis of amino acid esters in acetonitrile-water were unusually dependent on the solvent composition. The products obtained as precipitates in high concentrations of acetonitrile were L-amino acids of high optical purities, and effective optical resolution of amino acids was achieved.     

Reduced cholecystokinin in the brain of LEC rats with hepatic encephalopathy.

Levels of cholecystokinin (CCK) immunoreactivity and distribution of CCK immunoreactive cells were studied in the cerebral cortex of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy. CCK immunoreactivity in water extract of cerebral cortex of LEC rats with hepatic encephalopathy (n = 7) was 41.5 +/- 2.6 (mean +/- S.E.M. pmol/g wet wt.) and that of LEC rats without encephalopathy (n = 8) was 67.1 +/- 6.9, the difference being significant (P less than 0.01). CCK immunoreactive cells assessed by immunohistochemistry were also markedly decreased in the cortex of LEC rats with hepatic encephalopathy of stage IV. Thus, CCK reduction was observed in the cerebral cortex of LEC rats with hepatic encephalopathy which are provided as a model for analysis of the pathogenesis of acute hepatic encephalopathy.     

Endothelin: a new inhibitor of renin release

Endothelin is a recently-discovered vasoconstrictor peptide which is produced by endothelium and acts on vascular smooth muscle cells. At present its actions on other organs or cells are unknown. We studied the effect of endothelin on renin release in a dynamic superfusion system of dispersed rat juxtaglomerular (JG) cells. Endothelin in concentrations of 10(-11) M or more inhibited renin release dose-dependently and this inhibitory action vanished in the absence of extracellular Ca. It is suggested that endothelin is an inhibitory regulator of renin secretion from JG cells and its action is Ca-dependent.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.     

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

The effect of 2-deoxy-D-glucose on insulin release by neonatal rat B cells in culture

The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.     

Isolation and Purification of Cytochrome b561 from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ was removed from chromatophores of a photoanaerobicallygrown Rhodopseudomonas sphaeroides by deoxycholate-cholate andTriton X-100 treatments of the chromatophores. The cytochromewas purified by ammonium sulfate fractionation and gel filtration.Its molecular weight was 45,000 (45 kD) and it was composedof three subunits with molecular weights of 23 kD, 19 kD andless than 6 kD. The cytochrome preparation had absorption maximaat 414 nm in the oxidized form, and at 428, 530 and 561 nm inthe reduced form. Its pi was 4.8. The midpoint potential ofthis cytochrome was 153 mV at pH 7.0. The compound was autooxidizable,and it had cytochrome c oxidase activity. (Received May 16, 1983; Accepted September 8, 1983)