Biomedical subjects

M S Anderson

Publications and source records attributed to M S Anderson.

At least 37 records · Page 2Linked to original sources

PubMed1998

One-day enzymatic synthesis and purification of UDP-N- [1-14C]acetyl-glucosamine.

UDP-GlcN[1-14C]Ac was synthesized in a single enzymatic reaction from [1-14C]acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctional Escherichia coli GlmU protein. Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight. GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography. Several methods were developed for rapid purification of UDP-GlcN[1-14C]Ac: an HPLC method handled micromole (microcurie) loads. Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP-GlcN[1-14C]Ac. These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors.

ATP-Binding Cassette Transporters↗

PubMed1997

Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily.

Bacterial peptidoglycan biosynthesis includes four enzymatic reactions in which successive amino acid residues are ligated to uridine diphospho-N-acetylmuramic acid (UDP-MurNAc). By comparing the amino acid sequences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified. A profile search of Swissprot for related sequences revealed that these regional similarities were present in the folyl-gamma-polyglutamate ligases. These sequence homologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an acyl phosphate intermediate. Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alanyl-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188). All mutations were highly detrimental to activity with enzyme specific activity reductions of 200-4500-fold, validating the critical nature of these residues. DNA sequence analysis from three E. coli mutants harboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mutations revealed the presence of point mutation(s) closely associated with the fourth of these aligned regions. The murF2 allele, expressed and purified as a glutathione S-transferase::MurF2 fusion, was 181-fold less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C). Thus the murF2 temperature-sensitive phenotype arises from a point mutation within a highly conserved region within this protein family. These data argue that these proteins comprise a superfamily of three substrate amide ligases that share significant structural and catalytic homologies.

Amino Acid Sequence↗

PubMed1997

Withholding research results in academic life science. Evidence from a national survey of faculty.

OBJECTIVES: To identify the prevalence and determinants of data-withholding behaviors among academic life scientists. DESIGN: Mailed survey of 3394 life science faculty in the 50 universities that received the most funding from the National Institutes of Health in 1993. PARTICIPANTS: A total of 2167 faculty responded to the survey, a 64% response rate. OUTCOME MEASURES: Whether respondents delayed publication of their research results for more than 6 months and whether respondents refused to share research results with other university scientists in the last 3 years. RESULTS: A total of 410 respondents (19.8%) reported that publication of their research results had been delayed by more than 6 months at least once in the last 3 years to allow for patent application, to protect their scientific lead, to slow the dissemination of undesired results, to allow time to negotiate a patent, or to resolve disputes over the ownership of intellectual property. Also, 181 respondents (8.9%) reported refusing to share research results with other university scientists in the last 3 years. In multivariate analysis, participation in an academic-industry research relationship and engagement in the commercialization of university research were significantly associated with delays in publication. Odds ratios (ORs) and 95% confidence intervals (CIs) were 1.34 (1.07-1.59) and 3.15 (2.88-3.41), respectively. Variables associated with refusing to share results were conducting research similar to the Human Genome Project (OR, 2.09; 95% CI, 1.75-2.42), publication rate (OR, 1.02; 95% CI, 1.01-1.03), and engagement in commercialization of research (OR, 2.45; 95% CI, 2.08-2.82). CONCLUSIONS: Withholding of research results is not a widespread phenomenon among life-science researchers. However, withholding is more common among the most productive and entrepreneurial faculty. These results also suggest that data withholding has affected a significant number of life-science faculty and further study on data-withholding practices is suggested.

Biological Science Disciplines↗

PubMed1997

Mayer-Rokitansky-Kuster-Hauser syndrome diagnosed by magnetic resonance imaging in a 15-year-old girl.

STUDY OBJECTIVE: Mayer-Rokitansky-Kuster-Hauser syndrome diagnosed by magnetic resonance imaging (MRI) in a 15-year-old girl with primary amenorrhea is reported. DESIGN: The presentation, MRI, and the subsequent evaluation and treatment of an adolescent female patient with Rokitansky syndrome are described. Correlation is made with previous clinical, pathologic, and imaging reports in the literature. SETTING: An adolescent girl with primary amenorrhea was referred to our institution for completion of her diagnostic work-up. Previous limited evaluations suggested the presence of anomalies of the genitourinary tract. Further delineation of the suspected congenital defects was necessary. PARTICIPANT: The 15-year-old female patient was evaluated by the gynecology service. Diagnostic radiology and pediatric urology were consulted. INTERVENTIONS: MRI, physical examination under anesthesia, and cystoscopy were performed. After initial nonoperative treatment, the patient underwent hysterectomy and sigmoid vaginoplasty. MAIN OUTCOME MEASURES: The patient's primary amenorrhea was explained. Mayer-Rokitansky-Kuster-Hauser syndrome was diagnosed. Vaginal agenesis and widely separated rudimentary uterine horns were well shown by the MRI. Associated skeletal anomalies were noted. A treatment plan was initiated based on a good understanding of the anatomic defects. RESULTS: The MRI and physical examination firmly established the diagnosis. The patient was counseled and managed conservatively at first. Hysterectomy and vaginoplasty were subsequently performed. CONCLUSIONS: Mayer-Rokitansky-Kuster-Hauser syndrome is an unusual müllerian-duct anomaly that is a cause of primary amenorrhea. It can be confidently and noninvasively diagnosed with MRI. The MRI demonstration of vaginal, cervical, and uterine morphology contributes significantly to treatment planning and patient management.

Abnormalities, Multiple↗

PubMed1997

Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway.

The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis. Although present in all gram-negative bacteria examined, the deacetylase from E. coli is the only example of this enzyme that has been expressed and purified. In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment. The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E. coli protein and that possesses a nearly identical hydropathy profile. In order to verify function, we subcloned the P. aeruginosa lpxC gene into the T7-based expression vector pET11a. Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction. We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps. The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P. aeruginosa. In contrast, deacetylase activity in crude extracts of E. coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner. The lpxC gene has no significant homology with amidase signature sequences. Therefore, we assign this protein to the metalloamidase family as a member with a novel structure.

Amidohydrolases↗

PubMed1996

Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion.

The D-alanyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escherichia coli and expressed as a glutathione S-transferase (GST) fusion using the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one step by affinity chromatography. The purified fusion protein was strongly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered by changes in assay pH or by cleavage from the fusion partner. However, this effect was suppressed by the addition of 0.5 M NaCl. Initial velocity and dead-end inhibitor studies with the fusion enzyme were most consistent with a sequential ordered kinetic mechanism for the forward reaction in which ATP binds to free enzyme, followed by tripeptide and D-Ala-D-Ala in sequence prior to product release. Reported homologies between the MurF protein and the three preceding steps of cytoplasmic murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these enzymes will be found to proceed via this mechanism.

Adenosine Triphosphate↗

PubMed1996

Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Amidohydrolases↗

PubMed1996

Carbon source regulation of PIS1 gene expression in Saccharomyces cerevisiae involves the MCM1 gene and the two-component regulatory gene, SLN1.

The Saccharomyces cerevisiae PIS1 gene encodes phosphatidylinositol synthase. The amount of phosphatidylinositol synthase is not affected by the presence of inositol and choline in the growth medium. This is unusual because the amounts and/or activities of other phospholipid biosynthetic enzymes are affected by these precursors, and the promoter of the PIS1 gene contains a sequence resembling the regulatory element that coordinates the inositol-mediated regulation (UASINO). We found that transcription of the PIS1 gene was insensitive to inositol and choline and did not require the putative UASINO regulatory sequence or the cognate regulatory genes (INO2 and OPI1). The PIS1 promoter includes sequences (MCEs) that bind the Mcm1 protein. Because the Mcm1 protein interacts with both the Sln1 and the Gal11 regulatory proteins, we examined the effect of mutant alleles of the MCM1 and SLN1 genes and carbon source on expression of the PIS1 gene. We found that expression of the PIS1 gene was reduced when cells were grown in a medium containing glycerol and increased when grown in a medium containing galactose relative to cells grown in a glucose medium. The glycerol-mediated repression of PIS1 gene expression required both the MCM1 gene and the MCEs, whereas the SLN1 gene was required for full galactose-mediated induction of a PIS1-lacZ reporter gene. Thus, PIS1 gene expression is unique among the phospholipid biosynthetic structural genes because it is uncoupled from the inositol response and regulated in response to the carbon source. This is the first example in yeast of a complete circuit linking a stimulus (carbon source) to gene regulation (PIS1) using a two-component regulator (SLN1).

Base Sequence↗

PubMed1996

Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid a biosynthesis.

The first enzyme of lipid A assembly in Escherichia coli is an acyltransferase that attaches an R-3-hydroxymyristoyl moiety to UDP-GlcNAc at the GlcNAc 3-OH. This reaction is reversible and thermodynamically unfavorable. The subsequent deacetylation of the product, UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc, is therefore the first committed step of lipid A biosynthesis. We now demonstrate that inhibition of either the acyltransferase or the deacetylase in living cells results in a 5-10-fold increase in the specific activity of the deacetylase in extracts prepared from such cells. Five other enzymes of the lipid A pathway are not affected. The elevated specific activity of deacetylase observed in extracts of lipid A-depleted cells is not accompanied by a significant change in the Km for the substrate, but is mainly an effect on Vmax. Western blots demonstrate that more deacetylase protein is indeed made. However, deacetylase messenger RNA levels are not significantly altered. Inhibition of lipid A biosynthesis must either stimulate the translation of available mRNA or slow the turnover of pre-existing deacetylase. In contrast, inhibition of 3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis has no effect on deacetylase specific activity. The underacylated lipid A-like disaccharide precursors that accumulate during inhibition of Kdo formation may be sufficient to exert normal feedback control.

Amidohydrolases↗

PubMed1996

Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes.

BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. UDCA is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2+ agonist and stimulates biliary exocytosis and hepatocellular Ca2+ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. METHODS: The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accummulation of sn-1,2-diacylglycerol (DAG), the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. RESULTS: Immunoblotting studies showed the presence of four isoenzymes (alpha, delta, epsilon, and zeta). The phorbol ester phorbol 12-myristate 13-acetate (1 mumol/L) induced translocation of alpha-PKC, delta-PKC, and epsilon-PKC from cytosol to a particulate membrane fraction, a key step for activation of PKC. TUDCA, but not taurocholic acid, selectively induced translocation of the alpha-PKC isoenzyme from cytosol to the membranes. In addition, TUDCA induced a significant increase in hepatocellular DAG mass and stimulated membrane-associated PKC activity. CONCLUSIONS: TUDCA might stimulate Ca(2+)-dependent hepatocellular exocytosis into bile in part by activation of alpha-PKC.

Animals↗

PubMed1996

Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host.

Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.

Animals↗

PubMed1995

The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

Alleles↗

PubMed1995

Physical map locations of the phospholipid biosynthetic structural and regulatory genes of Saccharomyces cerevisiae.

Here we report the physical map locations of five genes required for phospholipid biosynthesis in Saccharomyces cerevisiae. These include four structural genes (INO1, CHO2, OPI3 and PIS1) and one global negative regulatory gene (UME6). Collectively, this information completes the mapping of all phospholipid biosynthetic structural and regulatory genes identified to date.

Chromosome Mapping↗

PubMed1994

An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis.

Escherichia coli strain SM101 harbors a temperature-sensitive allele (lpxA2) of the gene encoding UDP-Glc-NAc acyltransferase (the first enzyme of the lipid A pathway). SM101 is temperature-sensitive for lipid A biosynthesis and growth. To determine whether or not E. coli mutants lacking lipid A can be isolated, we examined temperature-resistant revertants of SM101. All regained the ability to synthesize lipid A. However, some were not true revertants but had acquired mutations in a neighboring gene (orf17), while retaining the original lpxA2 lesion. Cell extracts of such revertants displayed 2-5 fold reductions in the specific activity of (3R)-hydroxymyristoyl-ACP dehydrase. Wild-type cells that overproduced the protein encoded by orf17 overproduced (3R)-hydroxymyristoyl-ACP dehydrase activity as much as 170-fold, suggesting that orf17 is the structural gene for the dehydrase. The proposed function of orf17 is further supported by its sequence similarity to fabA, the structural gene for (3R)-hydroxydecanoyl dehydrase of E. coli. We suggest that bypass of the lpxA2 phenotype by mutations in orf17 may be due to an increased (3R)-hydroxymyristoyl-ACP pool. The orf17 gene (which we now designate fabZ) is not regulated by fadR. However, orf17 may be related to sefA, a suppressor of certain lesions in the cell division/lipid A biosynthesis gene, envA.

Amino Acid Sequence↗

PubMed1994

Enhancement of merocyanine 540 uptake and photodynamic cell killing by salicylates.

Salicylate and several structurally analogous compounds enhance merocyanine 540 (MC540)-photosensitized killing of leukemia cells (M. A. Anderson, B. Kalyanaraman, and J. B. Feix, Cancer Res., 53: 806-809, 1993). In this work, we show that salicylic acid enhances the binding of MC540 prior to illumination, as well as the light-stimulated uptake of MC540 by target L1210 murine and K562 human leukemia cells. Acetylsalicylic acid, 2,3- and 2,5-dihydroxybenzoic acids, and sodium benzoate also enhance MC540 uptake. The irradiation dose responses for loss of cell survival and enhanced MC540 uptake are well correlated, both being shifted to earlier time points in the presence of salicylate. Salicylic acid also enhanced photodynamic cell killing of A549 lung carcinoma and NIH:OVCAR-3 ovarian carcinoma cells, two cell types which are relatively resistant to MC540-mediated photosensitization. Cellular uptake of the anionic, potential-sensitive oxonol dye, bis-(1,3-dibutylbarbituric acid)-trimethine oxonol, is also increased by salicylate in a dose-dependent fashion. In contrast, cellular uptake of the cationic cyanine dye, 3,3'-dihexyloxacarbocyanine, is unaffected by salicylate. These studies suggest that increased uptake of MC540 is the basis of salicylate enhancement and that changes in plasma membrane potentials may play a mechanistic role in the potentiation of MC540 binding and cell killing.

Animals↗

PubMed1994

Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24.

Duchenne and Becker muscular dystrophies are caused by defects of dystrophin, which forms a part of the membrane cytoskeleton of specialized cells such as muscle. It has been previously shown that the dystrophin-associated protein A1 (59-kDa DAP) is actually a heterogeneous group of phosphorylated proteins consisting of an acidic (alpha-A1) and a distinct basic (beta-A1) component. Partial peptide sequence of the A1 complex purified from rabbit muscle permitted the design of oligonucleotide probes that were used to isolate a cDNA for one human isoform of A1. This cDNA encodes a basic A1 isoform that is distinct from the recently described syntrophins in Torpedo and mouse and is expressed in many tissues with at least five distinct mRNA species of 5.9, 4.8, 4.3, 3.1, and 1.5 kb. A comparison of our human cDNA sequence with the GenBank expressed sequence tag (EST) data base has identified a relative from human skeletal muscle, EST25263, which is probably a human homologue of the published mouse syntrophin 2. We have mapped the human basic component of A1 and EST25263 genes to chromosomes 8q23-24 and 16, respectively.

Amino Acid Sequence↗

PubMed1993

Enhanced antigen presentation in the absence of the invariant chain endosomal localization signal.

The cytosolic tail of the major histocompatibility complex class II-associated invariant chain (Ii) molecule is thought to contain the endosomal localization signal that directs and/or retains newly synthesized class II within the endosomal antigen processing compartment. To determine the role of this signal in class II transport and antigen presentation we have generated class II-positive L cell transfectants that coexpress wild type or truncated forms of Ii. Deletion of the endosomal localization signal from Ii results in rapid transport of class II-Ii complexes to the cell surface. Once at the cell surface, the complex is efficiently internalized, Ii is degraded, and class II free of Ii is recycled back to the plasma membrane. Interestingly, the truncated form of Ii is still able to increase the efficiency of antigen presentation to T cells. These data suggest that the ability of Ii to enhance antigen presentation is not limited to Golgi apparatus-endosomal sorting and raise the possibility that endocytosed class II can form immunogenic complexes with newly processed antigen.

Amino Acid Sequence↗

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↗