A re-examination of the interaction of vinculin with actin

Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined.     

Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase

The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P. R., Merrill, A. H., Jr., and Bell, R. M., (1981) J. Biol. Chem. 256, 11151-11159). Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine. Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure. Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation. The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.     

Primary structure of the cytoplasmic domain of human erythrocyte protein band 3. Comparison with its sequence in the mouse

We report here the peptide profile of the human cytoplasmic domain of band 3 protein (CDB-3). The peptide alignment was designed allowing for maximal homology with the murine protein whose sequence was deduced from cDNA analysis by Kopito and Lodish (Kopito, R.R., Anderson, M. and Lodish, H.F. (1987) J. Biol. Chem. 262, 8035-8040). In the human protein, part of the amino acid sequence has been determined by Kaul et al. (Kaul, R.K., Murthy, P.S.N., Reddy, A.G., Steck. T.L. and Kohler, H. (1983) J. Biol. Chem. 258, 7981-7990). We have sequenced most of the fragment not described by these author. The homology with the murine protein is high (90%), except in a few peptides where it is only 50%. The actual miniaturization of the techniques allows for the determination of a clear peptide profile of human CDB-3 starting from 10 ml blood samples. Our characterization of the peptide profile of membrane proteins is the first step towards the identification of genetic mutations, which have to be looked for in hemolytic anemia when the presence of an abnormal membrane protein is suspected.     

Creatine phosphate inhibition of heart lactate dehydrogenase and muscle pyruvate kinase is due to a contaminant

Previously reported inhibitions of heart lactate dehydrogenase (Guppy, M., and Hochachka, P.W. (1978) J. Biol. Chem. 253, 8465-8469) and muscle pyruvate kinase (Kemp, R.G. (1973) J. Biol. Chem. 248, 3963-3967) by creatine phosphate are due to oxalate which is a contaminant found in some commercial preparations of creatine phosphate.     

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.     

The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.     

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum

Phospholamban, a putative regulator of the Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum (SR), was purified from canine cardiac SR membranes. Cardiac SR was extracted with deoxycholate and fractionated with ammonium sulfate followed by gel permeation high performance liquid chromatography in the presence of the nonionic detergent, octa-ethylene glycol mono-n-dodecyl ether (C12E8), and KI. Further purification was achieved with CM-Sepharose CL 6B column chromatography in the presence of C12E8. The purified phospholamban showed a single band of 22,000 daltons on neutral sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412) and 27,000 daltons on alkaline SDS gels (Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685). Boiling of phospholamban in 2% SDS produced total conversion into the lower molecular weight component on SDS gels (11,000 on Laemmli gel and 10,500 on Weber and Osborn gel). The apparent molecular weight of phospholamban on SDS gels was slightly increased by cAMP-dependent phosphorylation. The extent of phosphorylation catalyzed by cAMP-dependent protein kinase in the purified phospholamban preparations was about 42 nmol of phosphate/mg of protein when the protein concentration was determined by the method of Lowry et al. (Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275), or 138 nmol/mg of protein based on the protein concentration estimated by the dye absorption method. Rabbit antisera were prepared against purified phospholamban. The obtained antisera were found to bind to purified phospholamban as well as that in cardiac SR. No reaction was detected in fast skeletal muscle SR by immunofluorescent staining of Western blots. The present preparation of purified phospholamban and the antisera should facilitate further understanding of the regulatory action of phospholamban on the calcium pump ATPase.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Characterization of the six chicken histone H1 proteins and alignment with their respective genes

Six histone H1 subtypes and histone H5, isolated from chicken erythrocyte nuclei, were visualized on acid/urea polyacrylamide gels. Four of the H1 subtypes have been purified to homogeneity by fast protein liquid chromatography on a strong cation exchange column. The other two subtypes were obtained as enriched fractions from the same fast protein liquid chromatography experiments. Because six chicken H1 genes have been completely sequenced (Coles, L.S., Robins, A. J., Madley, L.K., and Wells, J. R. E. (1987) J. Biol. Chem. 262, 9656-9663), it was possible to assign each of the six H1 proteins to a specific gene after amino acid sequence analysis of peptides derived from the subtypes.     

Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn

Epidermal growth factor stimulates a cyclic AMP-independent protein kinase associated with membrane vesicles derived from human epidermoid carcinoma cells (Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Biol. Chem. 254,. 4884-4891). The kinase specifically phosphorylates tyrosyl residues in a Mr = 150,000 membrane protein (Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255, 8363-8365). We show that the reverse reaction, catalyzed by a phosphotyrosyl-protein phosphatase associated with the membrane, is inhibited by Zn2+. Dephosphorylation of phosphotyrosyl residues in the Mr = 150,000 protein is completely inhibited by Zn2+ at concentrations as low as 10 microM, whereas other divalent cations have no substantial effect. Inhibition of the phosphatase was reversed by EDTA and the activity in membrane preparations was increased by EDTA or fluoride, agents commonly thought to be phosphatase inhibitors. Acid hydrolysis of the membrane proteins followed by analysis of phosphoamino acids by two-dimensional electrophoresis revealed that the phosphatase hydrolyzed phosphotyrosyl in preference to phosphoseryl residues. The specific inhibition of this phosphatase activity by low concentrations of Zn2+ may be indicative of the physiological importance of Zn2+ in the regulation of cellular phosphotyrosyl-protein levels.     

Mobile histone tails in nucleosomes. Assignments of mobile segments and investigations of their role in chromatin folding

The 13C NMR spectrum of isolated nucleosome core particles contains many sharp resonances, including resonances of alpha- and beta-carbons, indicating that certain terminal segments of histones rich in basic residues are highly mobile (Hilliard, R. R., Jr., Smith, R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 5992-5998). Specific histone termini can be removed sequentially from nucleosome core particles by mild treatment with alpha-chymotrypsin or chymotrypsin plus trypsin (Rosenberg, N. L., Smith. R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 12375-12383). Comparisons of the 13C NMR spectra of native and several partially proteolyzed core particles indicated that a minimum of residues 1-20 of H3 and 1-11 and 118-128 of H2a are contained in mobile segments of native cores. H4 did not appear to contribute to the resonances from mobile histone segments, but a possible contribution of H2b residues 1-16 could not be ruled out. The 13C NMR spectra of oligonucleosomes containing and lacking lysine-rich histones (H1, H5) were similar to each other and to that of native nucleosome cores both when the oligonucleosomes were in an extended conformation at low ionic strength and when they were in a more compact conformation at higher ionic strength. This similarity suggests that histones H1 and H5 must be largely immobilized upon chromatin binding and that the segments of core histones that are mobile in isolated nucleosome cores are not strongly bound to adjacent linker regions in intact chromatin, and are not immobilized by compaction to the degree achieved in 50 mM phosphate buffer.     

Identification of ammonium ion and 2,6-bis(omega-aminobutyl)- 3, 5-diiminopiperazine as endogenous factors that account for the "burst" of sphingosine upon changing the medium of J774 cells in culture

Cells in culture often undergo a "burst" of free sphingosine, sphingosine 1-phosphate, ceramide, and other bioactive lipids upon removal of "conditioned" medium, and at least one lipid signaling pathway (protein kinase C) has been shown to be affected by these changes (Smith, E. R. & Merrill A. H., Jr. (1995) J. Biol. Chem. 270, 18749-18758; Smith, E. R., Jones, P. L., Boss, J. M. & Merrill, A. H., Jr. (1997) J. Biol. Chem. 272, 5640-5646). Whereas increases in sphinganine and dihydroceramide are responses to provision of precursors for sphingolipid biosynthesis de novo in the new medium, the sphingosine burst is due to sphingolipid turnover upon removal of suppressive factor(s) in conditioned medium. This study describes the purification and characterization of these suppressive factors. Conditioned medium from J774 cells was fractionated into two components that suppress the burst as follows: ammonium ion, which reaches 2-3 mM within 48 h of cell culture; and a low molecular weight, cationic compound that has been assigned the structure 2, 6-bis(omega-aminobutyl)-3,5-diimino-piperazine (for which we suggest the name "batrachamine" based on its appearance) by (1)H and (13)C NMR, Fourier transform infrared spectroscopy, and mass spectrometric analyses. The physiological significance of these compounds as suppressors of sphingolipid metabolism is unclear; however, ammonium ion is a by-product of amino acid catabolism and reaches high concentrations in some tissues. Batrachamine is even more intriguing because this is, as far as we are aware, the first report of a naturally occurring compound of this structural type. Considering the many cell functions that are affected by sphingoid bases and their derivatives, the effects of NH(4) and batrachamine on sphingolipid metabolism may have important implications for cell regulation.     

Identification by photoaffinity labeling of fatty acid-binding protein as a potential warfarin receptor in rat liver

Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli

Escherichia coli mutants defective in the pgsB gene are phosphatidylglycerol-deficient in certain genetic settings and accumulate novel, glucosamine-derived phospholipids (Nishijima, M., and Raetz, C. R. H. (1979) J. Biol. Chem. 254, 7837-7844). The simplest of these compounds is 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) ("lipid X" of E. coli), in which beta-hydroxymyristoyl moieties are the sole fatty acid substituents (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report a sensitive radiochemical method for detection of 2,3-diacyl-GlcN-1-P in wild type E. coli and demonstrate that there are about 4000 molecules/cell (0.02% of the total CHCl3-soluble phosphorus). In mutants bearing the pgsB1 lesion, the levels are 100- to 300-fold higher. In addition, we have discovered a novel liponucleotide, UDP-2,3-diacyl-GlcN, that also accumulates in conjunction with the pgsb1 mutation. This material represents 0.005% of the wild type phospholipid and accumulates 50- to 100-fold in the mutant. The identification of UDP-2,3-diacyl-GlcN in E. coli is based on: 1) migration of a minor 32P-labeled lipid from wild type and mutant cells with a UDP-2,3-diacyl-GlCn standard during two-dimensional thin layer chromatography; 2) susceptibility of this 32P-labeled material to cleavage by a liponucleotide-specific pyrophosphatase; and 3) chromatographic identification of [32P]UMP and [32P]2,3-diacyl-GlcN-1-P (lipid X) as the sole products of the enzymatic degradation. As shown in the accompanying article, this novel nucleotide is crucial for biosynthesis of lipid A disaccharides in extracts of E. coli and Salmonella typhimurium.