Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.     

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.     

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres

The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.     

Isopentenoid synthesis in embryonic Drosophila cells: prenylated protein profile and prenyl group usage.

It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila Kc cells. [3H]Mevalonate was incorporated into 54 Kc cell peptides (18-92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C18 radio-HPLC tryptic digest profiles for delipidized, [3H]mevalonate-labeled (a) insect (Drosophila and Spodoptera frugiperda) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) Kc cell nuclear lamin, and (c) a 23.5-kDa purified Kc cell GTP-binding protein were compared and analyzed. [35S]Cysteine-labeled Kc cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [3H]mevalonate-labeled cells. A significant fraction (30-33%) of the doubly labeled tryptic peptides were eluted with greater than or equal to 93% acetonitrile. Kc cell nuclear lamin tryptic digests yielded a single 3H-labeled product which migrated as S-farnesylcysteine. The Kc cell 23.5-kDa GTP-binding protein's 3H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with greater than or equal to 93% acetonitrile. Moreover, the 3H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [3H]mevalonate-labeled Kc and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics.     

Structure of extended lipopolysaccharide glycoforms containing two globotriose units in Haemophilus influenzae serotype b strain RM7004

Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. Structural elucidation of the LPS from H. influenzae type b strain RM7004 was achieved by using electrospray ionization mass spectrometry (ESI-MS) and high-field NMR techniques on delipidated LPS and core oligosaccharide samples of LPS. It was found that the organism elaborates a series of related LPS glycoforms having a common inner-core structure, but differing in the number and position of attached hexose residues. LPS glycoforms containing between four and nine hexose residues were structurally characterized. The inner-core element was determined to be L-alpha-D-Hepp-(1-->2)-[PEA-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[P-->4]-alpha-KDOp-(2-->, a structural feature which has been identified in every H. influenzae strain investigated to date. Two major groups of isomeric glycoforms were characterized in which the terminal Hepp residue of the inner-core element was either substituted at the O-2 position with a beta-D-Galp residue or not. The structures of the major LPS glycoforms were found to have oligosaccharide chain extensions from O-3 of the middle Hepp residue. Glycoforms containing five and six hexose residues were most abundant and were shown to carry the tetrasaccharide unit alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->4)-alpha-D-Glcp at the O-3 position of the middle heptose. This tetrasaccharide displays the globoside trisaccharide (globotriose) as a terminal epitope, a structure that is found on many human cells (P(k) blood group antigen) and which is thought to be an important virulence determinant for H. influenzae. LPS glycoforms were characterized that had further chain extension from the beta-D-Glcp-(1--> residue of the proximal Hepp. In the fully extended LPS (Hex9/Hex8' glycoforms), both the proximal and middle heptose residues carried tetrasaccharide chains displaying terminal globotriose epitopes. In addition, the LPS was found to carry phosphorylcholine and O-acetyl groups.     

Fatty acylation of yeast glycoproteins proceeds independently of N-linked glycosylation

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild-type and asparagine-linked glycosylation-deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non-permissive temperature (37 degrees C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS-PAGE. In contrast, the wild-type yeast cells contained [3H]palmitate-labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS-PAGE. In addition, labeling of the wild-type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non-permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N-glycosylation in yeast cells.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis.

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b

We investigated the prevalence of antibodies in childrens' sera directed against outer membrane proteins (OMP) and fimbriae of Haemophilus influenzae type b. Invasive isolates of H. influenzae type b were enriched for fimbriae production; OMP and fimbriae were resolved by SDS-PAGE. After blotting to nitrocellulose, the proteins were incubated with homologous patient sera or with sera from healthy children. IgG antibodies bound to OMP were detected by immunoperoxidase staining. Immunoblotting was also performed using purified, nondenatured fimbriae as antigen. Nine of the 10 patients studied had antibodies in the acute serum directed against one or more of the OMP. Neither the acute nor the convalescent serum of the remaining patient contained antibodies against OMP. Antibodies against a greater number of OMP were present in the convalescent serum, in comparison to the acute serum, in 4 of the 10 patients. Five of 10 patients had antibodies against the purified fimbriae of an unrelated invasive isolate in either the acute or the convalescent serum. Acute sera from patients more frequently contained antibodies directed against OMP 60K (p less than or equal to 0.01) and OMP 51K (p less than or equal to 0.003) compared with the sera of healthy controls. In contrast, the sera of healthy children more frequently contained antibodies directed against OMP 40K (p less than or equal to 0.04). Sera from both patients and controls contained antibodies against commensal Haemophilus. We conclude that although antibodies against OMP are commonly present in healthy children, antibodies against certain OMP may be markers for susceptibility or protection.     

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.     

Effect of heparin on the utilization in vitro of labelled glycerides from triglyceride-rich lipoproteins in rat adipose tissue

Triglyceride-rich lipoproteins from adult rat plasma were labelled in vivo with 3H in the esterified fatty acids and 14C in the labelled glyceride glycerol of neutral lipids by injecting i.v. sodium 9-10 (n)-[3H] palmitate and [U-14C] glycerol, after which the prelabelled lipoproteins were purified by ultracentrifugation and dialysis. The lipoproteins were incubated in vitro, in the presence or not of heparin, with pieces of epididymal fat pads or isolated adipocytes from fed rats. The disappearance of both [3H]- and [14C] lipids from the media was greater when incubations were performed with adipocytes than with fat-pad pieces and it increased with heparin in both preparations. More 3H-label than 14C was found in the tissue lipids, a higher percentage being present in adipocytes than in fat-pad pieces, and the amount of label in tissue lipids was always enhanced by heparin. Some 14C-label appeared as esterified fatty acids in both tissue preparations and it also was enhanced by the presence of heparin. These findings are in agreement with the recognized influence of heparin on the release of lipoprotein lipase and show the direct relationship between heparin action and tissue ability to take up products of lipoprotein triglyceride breakdown. They also demonstrate the ability of adipose tissue to metabolize glycerol coming from the hydrolysis of lipoprotein glycerides.     

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of [3H] acetic, [3H]myristic, or [3H]palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH2 or COOH terminus.     

Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D.

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.