Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben (Glycine max).Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p-coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism.The increases in all of these enzyme activities could be further enhanced by illunination of the cells.No comparable light effects and no significant changes were observed for the specific activity of an S-adenosylmethionine:o-dihydric phenol m-O-mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.     

Enzymatic methylations: III. Cadaverine-induced conformational changes of E.coli tRNAfMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation+

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.     

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000+/-2000 in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca(2+) and is adsorbed to BaSO(4). The pK(a) of its BaSO(4)-binding group(s) is 3.1-3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO(4) and, since the ability of the whole protein to bind to BaSO(4) is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.     

Some measures of information arising in statistical games

This paper discusses some measures of information which naturally arise in the context of statistical games (games against nature). Some useful inequalities are proven relating the entropy to the value of information provided by experiments. Two other measures, based on the notion of a metric as informational distance and that of a diameter value are also discussed.     

Regulation of the pentose phosphate cycle

1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Oxidation of omega-hydroxylated fatty acids and steroids by alcohol dehydrogenase

Recrystallized alcohol dehydrogenase from horse liver was found to oxidize 17-hydroxystearic acid into 17-oxostearic acid, the 17-L-enantiomer faster than the 17-D-enantiomer. Alone at high pH or in combination with aldehyde dehydrogenase, the alcohol dehydrogenase also catalyzed conversion of 18-hydroxystearic acid into 1, 18-octadecadioic acid and 5β-cholestane-3α,7α,12α,26-tetrol into 3α,7α,12α-trihydroxy-5β-cholestanoic acid. All the activities as well as the ethanol dehydrogenase activity disappeared after specific carboxymethylation of a single cystein residue at the active site of alcohol dehydrogenase. These results conclusively show that alcohol dehydrogenase itself has ω-hydroxyfatty acid dehydrogenase activity and ω-hydroxysteroid dehydrogenase activity.