Structure-based design of inhibitors of human L-xylulose reductase modelled into the active site of the enzyme

The program GRID was used to design potential inhibitors of human L-xylulose reductase based on a model of the holoenzyme in complex with n-butyric acid. The inclusion of phosphate or carboxylate functional groups in the ligand suggested an increase in the net binding energy of the complex up to 2.8- and 4.0-fold, respectively. This study may be useful in the development of potent and specific inhibitors of the enzyme.     

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.     

Volatile components in cell suspension cultures of Cryptomeria japonica

The cell suspension culture of Cryptomeria japonica contains volatile oils, the yield of which was 0.005–0.01% of the fresh cells. In the volatiles, five aldehydes, ten fatty acids and their esters, and two diterpenes of abietatriene and ferruginol have been found. Of these, palmitic acid is present as the most predominant component, amounting to ca 40% of the volatiles.     

Structure-specific effects of thyroxine analogs on human liver 3 alpha-hydroxysteroid dehydrogenase

The NADP(H)-linked oxidoreductase activity of a major isozyme of human liver 3 alpha-hydroxysteroid dehydrogenase was activated 5-, 4-, and 2-fold by D-thyroxine (T(4)), L-T(4) and DL-3,3', 5'-triiodothyronine (reverse T(3)), respectively. Kinetic analysis of the activation indicated that D-T(4), L-T(4), and reverse T(3) are non-essential activators, showing binding constants of 1.5, 1.1, and 3.6 microM, respectively. Comparison of the effects of the T(4) analogs on the activities of the mutant enzymes suggests that the binding site is composed of at least Lys-270, Arg-276, and the C-terminal loop of the enzyme. L-T(3), DL-thyronine, and D-tyrosine had no effect on the enzyme, but 3,5,3',5'-tetra- and 3,5, 3'-tri-iodo thyropropionic acids were potent competitive inhibitors with K(i) values of 42 and 60 nM, respectively, with respect to the substrate. The inhibition constant was lowered upon the activation of the enzyme by D-T(4), and the inhibition by the deamino derivatives of T(4) and T(3) disappeared upon modification of the C-terminal loop of the enzyme, but not upon replacement of Lys-270 or Arg-276 with Met. These results indicate that, depending on their structures, the T(4) analogs bind differently to two distinct sites at the active center of the enzyme to produce stimulatory and inhibitory effects.     

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Identity of dimeric dihydrodiol dehydrogenase as NADP(+)-dependent D-xylose dehydrogenase in pig liver

Dimeric dihydrodiol dehydrogenases (DDs, EC 1.3.1.20), which oxidize trans-dihydrodiols of aromatic hydrocarbons to the corresponding catechols, have been molecularly cloned from human intestine, monkey kidney, pig liver, dog liver, and rabbit lens. A comparison of the sequences with the DNA sequences in databases suggested that dimeric DDs constitute a novel protein family with 20 gene products. In addition, it was found that dimeric DD oxidizes several pentoses and hexoses, and the specificity resembles that of NADP(+)-dependent D-xylose dehydrogenase (EC 1.1.1.179) of pig liver. The inhibition of D-xylose dehydrogenase activity in the extracts of monkey kidney, dog liver and pig liver, its co-purification with dimeric DD activity from pig liver, and kinetic analysis of the D-xylose reduction by pig dimeric DD indicated that the two enzymes are the same protein.     

Activity of starch synthase and the amylose content in rice endosperm

The content of amylose in endosperm of non-waxy japonica rice (Oryza sativa cv Akitakomachi) was increased by lowering the growth temperature from 25° to 15° during the ripening period. The activities of sucrose synthase, ADPglucose pyrophosphorylase, starch branching enzyme (Q-enzyme) and soluble starch synthase in endosperm developed at 15° were lower than or similar to those at 25°, when compared on a endosperm basis at the similar ripening stage. In contrast, the activity of starch granule-bound starch synthase, which is considered to be indispensable for amylose synthesis, was higher by 3–3.5-fold in the endosperm developed at the low temperature than that at the high ambient temperature. The results suggest that the low temperature specifically accelerates the expression of the bound starch synthase gene (waxy gene) in rice endosperm, which resulted in elevated amylose biosynthesis in the endosperm when developed at lower temperatures.     

Purification and Characterization of an Aminopeptidase, LPAase 2, from Euonymus Leaves

An aminopeptidase, LPAase 2, from the leaves of Euonymus alatusf. ciliato-dentatus was purified about 240-fold by a combinationof DEAE-cellulose and Sephadex G-100 column chromatographies.The molecular weight of LPAase 2 was estimated to be about 62,000,and the optimum pH for the hydrolytic activity against leucinep-nitroanilide(LPA) was 7.6. LPAase 2 hydrolyzed LPA, leucine-rß-naphthylamide(leucine-NA), phenylalanine-NA and tyrosine-NA. It was inhibitedstrongly by p-chloromercuribenzoate (PCMB), iodoacetic acidand heavy metal ions, but was not affected by thiol compoundsand metal-chelating reagents. Therefore, a sulfhydryl groupcould be involved in the active site of LPAase 2. None of themetal ions tested promoted LPAase 2 activity. The propertiesof LPAase 2 were compared with those of aminopeptidases reportedfor other plants. (Received November 24, 1983; Accepted April 16, 1984)     

Nucleotide sequence of valine tRNA mo5UAC from bacillus subtilis.

A valine tRNA was purified from Bacillus subtilis W168 by a combined use of several column chromatographic systems. The nucleotide sequence was determined to be pG-G-A-G-G-A-U-U-A-G-C-U-C-A-G-C-D-G-G-G-A-G-A-G-C-A-U-C--G-C-C-U-mo5U-A-C-m6-A -A-G-C-A-G-A-G-G-m7G-U-C-G-G-C-G-G-T-psi-C-G-A-G-C-C-C-G-U-C-A-U-C-C-U-C-C-A-C- C-AOH with the main use of of non-labeled tRNA and with the subsidiary use of [32P]-post-labeled sample. This tRNA contains 5-methoxy-uridine (mo5U) at the wobble position of the anticodon. A binding experiment of valyl-tRNA to ribosome revealed that mo5U is recognized by A and G, and fairly well by U.