PubMed Health⌕ Search

Biomedical subjects

C R Raetz

Publications and source records attributed to C R Raetz.

At least 73 records · Page 4Linked to original sources
PubMed1994

Genetic evidence supporting the role of peroxisome assembly factor (PAF)-1 in peroxisome biogenesis. Polymerase chain reaction detection of a missense mutation in PAF-1 of Chinese hamster ovary cells.

The peroxisome/plasmalogen-deficient Chinese hamster ovary (CHO) mutant cell line ZR-78.1 contains a missense mutation in its cDNA-encoding peroxisome assembly factor-1 (PAF-1). Using a rapid polymerase chain reaction assay, we now demonstrate that the genome of ZR-78.1 contains only the mutant allele. When mutant ZR-78.1 is fused with wild-type karyoplasts, occasional "negative nuclear hybrids" are observed that lack peroxisomes (Allen, L.-A. H., Morand, O. H., and Raetz, C. R. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7012-7016). Despite the fact that negative nuclear hybrids are tetraploid, they do not contain the wild-type PAF-1 gene, suggesting that a chromosome fragment bearing the wild-type copy of PAF-1 was lost. Negative nuclear hybrids reconstituted with wild-type cytoplasts do contain a wild-type PAF-1 gene, indicating that the cytoplasts somehow reintroduced the wild-type PAF-1 allele without increasing ploidy. These findings support the role of PAF-1 and exclude the hypothesis of an additional cytoplasmic requirement for reinitiation of peroxisome biogenesis in peroxisome-deficient CHO cells. The plasmalogen deficiency and some other biochemical properties of ZR-78.1 are partially corrected in 5-azacytidine-treated subclones. However, such pseudo-revertants do not contain peroxisomes, consistent with the fact that there is no wild-type PAF-1 gene to reactivate by demethylation.

Acyltransferases↗
PubMed1994

Biosynthesis of a structurally novel lipid A in Rhizobium leguminosarum: identification and characterization of six metabolic steps leading from UDP-GlcNAc to 3-deoxy-D-manno-2-octulosonic acid2-lipid IVA.

Lipopolysaccharides (LPSs) are prominent structural components of the outer membranes of gram-negative bacteria. In Rhizobium spp. LPS functions as a determinant of the nitrogen-fixing symbiosis with legumes. LPS is anchored to the outer surface of the outer membrane by the lipid A moiety, the principal lipid component of the outer bacterial surface. Several notable structural differences exist between the lipid A of Escherichia coli and that of Rhizobium leguminosarum, suggesting that diverse biosynthetic pathways may also exist. These differences include the lack of phosphate groups and the presence of a 4'-linked GalA residue in the latter. However, we now show that UDP-GlcNAc plays a key role in the biosynthesis of lipid A in R. leguminosarum, as it does in E. coli. 32P-labeled monosaccharide and disaccharide lipid A intermediates from E. coli were isolated and tested as substrates in cell extracts of R. leguminosarum biovars phaseoli and viciae. Six enzymes that catalyze the early steps of E. coli lipid A biosynthesis were also present in extracts of R. leguminosarum. Our results show that all the enzymes of the pathway leading to the formation of the intermediate 3-deoxy-D-manno-2-octulosonic acid (Kdo2)-lipid IVA are functional in both R. leguminosarum biovars. These enzymes include (i) UDP-GlcNAc 3-O-acyltransferase; (ii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase; (iii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcN N-acyltransferase; (iv) disaccharide synthase; (v) 4'-kinase; and (vi) Kdo transferase. Our data suggest that the early steps in lipid A biosynthesis are conserved and that the divergence leading to rhizobial lipid A may occur at a later stage in the pathway, presumably after the attachment of the Kdo residues.

Acetates↗
PubMed1994

Endotoxin biosynthesis in Pseudomonas aeruginosa: enzymatic incorporation of laurate before 3-deoxy-D-manno-octulosonate.

Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria.

ADP Ribose Transferases↗
PubMed1993

UDP-N-acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin biosynthesis is thermodynamically unfavorable.

UDP-N-acetylglucosamine acyltransferase of Escherichia coli catalyzes the reaction, UDP-GlcNAc + R-3-hydroxymyristoyl-ACP--> UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + ACP. Using Matrex Gel Green A and heparin-agarose, we have purified the enzyme to near homogeneity from a strain that overproduces it 474-fold. The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with results of previous radiolabeling experiments in mini-cells. The amino-terminal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA sequencing (Coleman, J., and Raetz, C. R. H. (1988) J. Bacteriol. 170, 1268-1274). At saturating concentrations of the second substrate, the apparent Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 microM, respectively. There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 microM) but is inactive (< 0.01%) as an alternate substrate. The most remarkable feature of the reaction is its unfavorable equilibrium constant, Keq approximately equal to 0.01, which is not predicted by model S-->O acyl transfer reactions. Thus, although UDP-GlcNAc acyltransferase catalyzes the first unique step of lipid A biosynthesis, it is the second enzyme (the deacetylase) that commits the substrates to this pathway. The specific activity of the deacetylase is elevated approximately 5-fold when lipid A synthesis is inhibited.

Acyltransferases↗
PubMed1993

The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis.

The possibility that the firA gene of Escherichia coli (Dicker, I. B., and Seetharam, S. (1991) Mol. Microbiol. 6, 817-823) might function in lipid A biosynthesis was examined based on its homology to the lpxA gene, which encodes UDP-N-acetylglucosamine O-acyl-transferase, the first enzyme in lipid A formation. Extracts of a temperature-sensitive firA mutant, RL-25, were assayed for their ability to acylate UDP-GlcNAc, using a coupled assay. The results suggested that extracts of RL-25 might be defective in the third enzyme of this pathway, the UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. Living cells of RL-25 also displayed a 5-fold decreased rate of lipid A biosynthesis at the nonpermissive temperature as judged by a 32Pi incorporation assay. In order to examine N-acyltransferase activity directly, the substrate [alpha-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcN was synthesized enzymatically. N-Acyltransferase specific activity in RL-25 extracts was reduced to less than 10% of wild-type. When the wild-type firA gene was cloned into a T7-based expression vector, N-acyltransferase specific activity increased almost 360-fold relative to wild-type extracts, demonstrating that firA is the structural gene for the enzyme. The N-acyltransferase displays absolute specificity for the R-3-OH moiety of R-3-hydroxymyristoyl-ACP, as does the O-acyltransferase, consistent with the placement of R-3-hydroxymyristate in E. coli lipid A.

Acyltransferases↗
PubMed1993

Peroxisome-deficient Chinese hamster ovary cells with point mutations in peroxisome assembly factor-1.

Chinese hamster ovary (CHO) mutant cells deficient in peroxisome biogenesis regain peroxisomes after transfection with a cDNA coding for peroxisome assembly factor (PAF)-1 from rat liver. Reconstitution of the transfected mutant cells with wild-type cytoplasm was not required, demonstrating that expression of the PAF-1 gene alone was sufficient for the restoration of peroxisome biogenesis. Plasmalogen biosynthesis in the transfected mutants was also restored to approximately wild-type levels. The nucleotide sequence of the cDNA encoding the open reading frame for PAF-1 from CHO-K1 cells was determined. This allowed us to identify point mutations of PAF-1 in two peroxisomal mutant cell lines. The mutation in ZR-78 cells changed a cysteine to a tyrosine codon in a region located at the carboxyl terminus of the protein, which resembles the zinc finger motif of DNA-binding proteins. A point mutation in the PAF-1 gene of ZR-82 leads to premature termination.

Amino Acid Sequence↗
PubMed1992

Identification and selective inhibition of an isozyme of steroid 5 alpha-reductase in human scalp.

Steroid 5 alpha-reductase (EC 1.3.1.22) catalyzes the reduction of testosterone to dihydrotestosterone. The 5 alpha-reductase found in human scalp has been compared with the enzyme found in prostate. The scalp reductase has a broad pH optimum centered at pH 7.0. This is distinctly different from the pH optimum of 5.5 observed with the prostatic form of the enzyme. These enzymes also differ in the Km for testosterone, which is 25-fold higher for the scalp reductase. The most significant difference between the two enzymes is their affinity for inhibitors. Two 4-azasteroids and a 3-carboxyandrostadiene are potent inhibitors of the prostatic reductase but are weak inhibitors of the scalp reductase. In contrast, several N-4-methylazasteroids are good inhibitors of the scalp reductase. These findings support a proposal that different isozymes of 5 alpha-reductase may exist in scalp and prostate. The scalp reductase was also compared to 5 alpha-reductase 1, one of the two enzymes recently cloned from human prostate [Andersson, S. & Russell, D. W. (1990) Proc. Natl. Acad. Sci. USA 87, 3640-3644; and Andersson, S., Berman, D. M., Jenkins, E. P. & Russell, D. W. (1991) Nature (London) 354, 159-161]. The characteristics of the cloned reductase 1 are comparable to those of the scalp reductase.

Animals↗
PubMed1992

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

A novel 3-deoxy-D-manno-octulosonic acid transferase from Chlamydia trachomatis required for expression of the genus-specific epitope.

DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced amino acid sequence of gseA shows 23% identity (66% similarity) to kdtA, an E. coli gene that codes for a bifunctional enzyme catalyzing the addition of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A precursors (Clementz, T., and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 9687-9696). Extracts of E. coli expressing gseA transfer at least one additional Kdo unit from CMP-Kdo to precursors already bearing the two Kdo residues attached by the kdtA gene product. Introduction of gseA into an E. coli mutant with a thermolabile kdtA gene product endows cell extracts with the ability to transfer not only the third but also the first two Kdos to lipid A precursors, demonstrating that the C. trachomatis enzyme is at least trifunctional. Given the similarities of these two Kdo transferases and the essentiality of Kdo in Gram-negative bacteria, lipopolysaccharide biosynthesis may be a target for development of novel drugs effective against chlamydiae.

Amino Acid Sequence↗
PubMed1992

The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide.

We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.

Amino Acid Sequence↗
PubMed1992

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.

Acyltransferases↗
PubMed1992

Increased cholesterol synthesis in Chinese hamster ovary cells deficient in peroxisomes.

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H., Bos, K., Raetz, C.R.H. and Wirtz, K.W.A. (1990) J. Biol. Chem. 265, 4105-4110). The consequences of the absence of peroxisomes and of nsL-TP on intracellular cholesterol metabolism have been investigated in two peroxisome-deficient CHO cell lines (CHO-82 and CHO-78). Compared with wild-type cells (CHO-K1), the incorporation of [3H]acetate into cholesterol was 3-fold higher in the CHO-82 cells and 2-fold higher in the CHO-78 cells. In agreement with an increased synthesis of cholesterol, a 2-3-fold higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was measured in both mutant cell lines. On the other hand, addition of low density lipoprotein (LDL), mevalonate (30 mM) or 25-hydroxycholesterol (2 micrograms/ml) to cells grown in lipoprotein-deficient serum, demonstrated that in both mutant cell lines the down-regulation of HMG-CoA reductase and of cholesterol synthesis were comparable to that in wild-type cells. These results strongly suggest that, in addition to down-regulation by LDL-derived cholesterol, mevalonate and 25-hydroxycholesterol, HMG-CoA reductase activity is under control of peroxisomes and/or nsL-TP.

Acetates↗
PubMed1992

Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.

Base Sequence↗