Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.     

Development of E.coli virus T1: the pattern of gene expression.

Summary T1 infected bacteria exhibit a distinct pattern of gene expression. The control of this expression is accessible to biochemical analysis. T1 induces the synthesis of 31 proteins inE. coli. The virion contains 15 proteins. By means of T1 amber mutants, 10 gene products have been assigned to specific T1 genes. Three classes of T1 proteins are defined by the kinetics of their syntheses: early, early-late and late proteins. The regulation of protein synthesis involves at least three mechanisms: for cessation of host gene expression, for discontinuation of the early class during the late phase and for induction of the late T1 proteins. The positive control of late gene expression is not coupled to replication. The host RNA-polymerase transcribes the viral genome throughout the infectious cycle. No virus coded RNA-polymerase is induced.     

Structural abnormalities in chromosome 15 in cell lines with reduced expression of beta-2 microglobulin

Using somatic cell hybrids, the gene for beta-2 microglobulin has been assigned to human chromosome 15. We found it of interest to study a number of human lymphoid cell lines in light of this finding, to analyze whether spontaneously occurring loss or reduction of beta-2 microglobulin could be correlated with any aberration in chromosome 15. The Daudi cell line was shown to be devoid of any beta-2 microglobulin in total extracts. Chromosome analysis showed that one of the two chromosomes 15 was deleted in the region q14q21 on the long arm; in some metaphases, both chromosomes were deleted in this region. The K562 cell line was found to express very low (if any) membrane-associated beta-2 microglobulin. Chromosome analysis showed that this line was near-triploid, with two normal chromosomes 15 and one translocation chromosome t(15;18) involving the long arm of chromosome 15, whereby the segment proximal to the breakage point in band q15 was lost. The Namalwa cell line showed a reduction in membrane-associated and total beta-2 microglobulin. Chromosome analysis showed this line to contain one chromosome 15 which was shorter than its normal homolog. The deletion could be identified as such in the region q14q21 in Daudi cells, but is probably somewhat smaller than the one in Daudi cells. Since analyses of beta-2 microglobulin production and chromosomes 15 on several other human cells failed to reveal any abnormalities in either of these respects, we postulate that genes responsible for beta-2 microglobulin synthesis and membrane expression could be located in the region q14q21 on the long arm of chromosome 15. Since beta-2 microglobulin associated with the membrane was found to be absent in the K562 line, where total beta-2 microglobulin was nearly as high as in cell lines with normal membrane expression, it is suggested that membrane expression of beta-2 microglobulin can be regulated independentlyAbbreviations used in this paper are BSS Earle's balanced salt solution - Q banding bands obtained by fluorescence with Quinacrine Mustard - R banding bands obtained by fluorescence with Acridine Orange - FITC fluorescein isothiocyanate - EBV Epstein-Barr virus     

Hypoxanthine-guanine phosphoribosyl transferase with altered substrate affinity in mutant mouse lymphoma cells

Cells with altered hypoxanthine-guanine phosphoribosyl transferase (HPRT) (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) have been selected. Compared to wild type, mutant enzyme has a reduced affinity for the substrate phosphoribosyl pyrophosphate and is more labile to heat inactivation. Mutant cells are resistant to 6-thioguanine at 33-39 degrees C and sensitive to hypoxanthine-aminopterin-thymidine at 37-39 degrees C, but not at 33 degrees C. We hypothesize that a single structural mutation of HPRT can explain these results.     

Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

Pericentric inversion in chromosome No.2 as a de novo mutation

Summary A patient with a de novo inversion of chromosome 2 is described. Two of her three children have the same inversion.     

In vivo inactivation of soluble hydrogenase of Alcaligenes eutrophus

The soluble, NAD⁺-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available. The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme. Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide. The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30° C was 1.5 h. The process of inactivation did not require ATP-synthesis. There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein. The possibility is discussed that the inactivation is due to superoxide radical anions (O ₂ ^- ) produced by the hydrogenase itself.     

Serum concentrations of vitamin D-binding protein (Group-specific component) in cystic fibrosis

Summary Vitamin D-binding protein (DBP) concentrations were determined in the sera of 90 cystic fibrosis homozygotes, 57 obligate heterozygotes, and 46 normal controls. Very significantly lower mean concentrations were found in the sera of CF homozygotes compared with both heterozygotes and controls (P<0.01, Wilcoxon Rank Sums Test). Subdivision of the samples by Gc phenotype showed that this relationship held true both in the Gc1 and Gc2-1 phenotypes. The small sample size of the Gc2 genotype makes the significance levels of limited usefulness, but the pattern of variation of DBP levels among CF homozygotes, heterozygotes, and controls was consistent with that observed for the Gc1 and Gc2-1 classes. Haptoglobin levels showed high coefficients of variation when compared among CF homozygotes, obligate heterozygotes, and controls, presumably because of nonspecific elevation in the acute-phase response. Alpha₂-macroglobulin levels were, if anything, slightly elevated in CF homozygotes compared with controls, while albumin levels showed no significant mean differences between these groups. Since the DBP concentration does not vary with age nor with levels of vitamin D and its metabolites, we interpret our results to mean that DBP levels are specifically decreased in cystic fibrosis, perhaps as the result of impaired glycosylation of the protein.A preliminary report of this work appeared in the Proceedings of the 8th International Congress on Cystic Fibrosis.     

Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus.

Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.     

Haplotype distribution and mutations at the PAH locus in Croatia

Summary Restriction fragment length polymorphism (RFLP) haplotypes and mutations at the phenylalanine hydroxylase (PAH) locus have been studied in 25 unrelated families from Croatia. The results of RFLP analysis demonstrated that 80% of the mutant alleles were associated with three haplotypes (1, 2 and 4). Eight mutations were detected on the background of six mutant haplotypes, comprising 68% of phenylketonuria (PKU) alleles in Croatia. The mutation in codon 408 was most frequent, as was the haplotype 2 allele with which it was associated. These data are in accordance with formerly published population genetic analyses at the PAH locus, and with studies revealing the molecular basis of the phenotypic heterogeneity of PKU. The codon 281 mutation was more frequent in Croatia than previously observed in other populations.