Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

Synthesis of bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by stepwise and selective formation of three disulfide Bridges

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Isolation and functional expression of pituitary peptidylglycine alpha-amidating enzyme mRNA. A variant lacking the transmembrane domain

We demonstrate that, in rat pituitary, peptidylglycine alpha-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.     

Cysteine proteinase inhibitor in the ascitic fluid of sarcoma 180 tumor-bearing mice is a low molecular weight kininogen. Partial NH2- and COOH-terminal sequences and susceptibility to various glandular kallikreins

It has been proposed that a cysteine proteinase inhibitor (CPI) found in the ascitic fluid of Sarcoma 180 tumor-bearing mice is a kind of kininogen (Itoh, N., Yokota, S., Takagishi, U., Hatta, A., and Okamaoto, H. (1987) Cancer Res. 47, 5560-5565). The first 40 NH2-terminal residues and 54 residues of the COOH-terminal sequence, including the bradykinin moiety of highly purified ascites CPI, were determined and compared with those of mammalian low molecular weight kininogens (LMWK). The significant identity between these amino acid sequences with those of other mammalian LMWKs suggests that ascites CPI corresponds precisely to mouse LMWK. This kininogen has a light chain composed of 43 amino acid residues, which contains a unique Met-Ala-Arg-bradykinin sequence. Hydroxyproline, which was recently identified in the bradykinin sequence of kininogen from the ascitic fluid of a cancer patient, was not found in the kinin moiety of this mouse kininogen. Among purified glandular kallikreins from human, hog, rat, and mouse, only mouse submaxillary gland kallikrein was able to release bradykinin from this kininogen. Kinetic studies using a newly synthesized fluorogenic substrate, N-t-butoxycarbonyl-Met-Ala-Arg-MCA, revealed that mouse kallikrein hydrolyzes this substrate approximately 80-fold faster than does hog kallikrein, suggesting that the unique Met-Ala-Arg-bradykinin sequence is responsible for the varied susceptibility of mouse kininogen to different kallikreins.     

Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI     

“Active” conformation of an inactive semi-synthetic ribonuclease-S

We have studied the integrity of folded structure of a fully active semi-synthetic ribonuclease-S which lacks amino acid residues 16 through 20, and an inactive one with the same residues deleted and 4-fluoro-l-histidine substituted for active site histidine 12. Using “Y” form crystals, we obtained X-ray structural data to a resolution of 2·6 Å and, incorporating phase information calculated from refined ribonuclease-S coordinates, prepared several types of electron density maps. These showed that the overall backbone structure and active site configuration of both analogues do not differ noticeably from those of the native protein. Structural homology extends to the catalytically relevant side-chain at position 12; 4-F-His² assumes the same position as does His in active ribonuclease-S. This supports the view that the 4-F-Hisl2 analogue is inactive due to a change in histidine 12 imidazole basicity, rather than to any significant conformational distortion within the active site.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Biosynthesis and excretion of hydrolases in germinating cereal seeds

The initial formation site of hydrolases in germinating cerealseeds and their subsequent release were examined using the substrate-filmtechnique. Early in the germination of cereal seeds, e.g., barley,wheat, rye, oat and maize, -amylase invariably appeared in theregion of the epithelial cells of the scutellum, whereas itlater gradually diffused into the entire region of the endospermtissues. The initial formation site of proteinase and RNA-asein germinating barley seeds was also confirmed to be in theepithelium. We conclude that the epithelium has a more importantrole in the enzymic breakdown of reserve substances stored inthe endosperm tissues than the aleurone layer. (Received October 19, 1979; )     

Continuous culture of reuber hepatoma cells in serum-free,arginine-, glutamine-and tyrosine-deprived chemically defined medium

Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic “marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle. This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry of Education, Science and Culture, Japan.