Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and GlcAβ1-3Gal(4-O-sulfate)β1-3Galβ1-4Xylβ1-O-Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with α-N-acetylgalactosaminidase and β-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was α-linked, as in the product previously synthesized using serum enzymes, and not β-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAβ1-4GlcNAcα1-4]2-GlcAβ1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Dipyridamole as an inhibitor of vaccinia virus replication

Dipyridamole in concentration of 25 microM inhibited the multiplication of vaccinia virus in about 90% of cells. In the presence of this substance, [3H]-uptake was sharply reduced both in uninfected and infected RK13 cells, while [14C]-uptake was not inhibited incorporation of [3H]-thymidine and [14C]-amino acids into viral particles. The present findings suggest that the antiviral character of dipyridamole is related with the inhibition of the substrate transport through the cell membrane.     

Polysaccharides with sulfate groups are human T-cell mitogens and murine polyclonal B-cell activators (PBAs). I. Fucoidan and heparin

We reported previously that dextran sulfate and carrageenan (kappa, lambda, and iota), which are sulfated polysaccharides, were human T-cell mitogens and mouse polyclonal B-cell activators (PBA). To clarify our working hypothesis further, we used fucoidan and heparin, both sulfated polysaccharides. The following results were obtained: (1) fucoidan is human T-cell mitogen and a mouse PBA; (2) heparin is also a human T-cell mitogen and a mouse PBA, but the degree of the responses by heparin is lower than that by fucoidan; (3) helper T-cell-dependent B-cell differentiation was not observed, since both fucoidan and heparin activate OKT4⁺ cells and OKT8⁺ cells nonspecifically and suppressor T cells (OKT8⁺ cells) may inhibit the helper function of B-cell differentiation by helper T cells (OKT4⁺ cells); and (4) our working hypothesis that polysaccharides with sulfate groups are human T-cell mitogens and mouse PBAs was further strengthened. The relationship between molecular weight and sulfate groups of the polysaccharides is discussed in detail.     

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

Pulsatile secretion of bioactive luteinizing hormone in adult male rhesus macaques: acute and chronic effects of orchidectomy

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.     

Effect of heparin on proliferation and signalling in BC3H-1 muscle cells. Evidence for specific binding sites

We studied binding and growth inhibitory properties of different glycosaminoglycans in growing and differentiated BC3H-1 muscle cells. Heparin (10 micrograms/ml) and heparan sulfate (10 micrograms/ml) significantly inhibited DNA synthesis in growing and differentiated cells, as monitored by [3H]thymidine incorporation. Binding of heparin to BC3H-1 cells was specific and time-dependent. Heparan sulfate was the only glycosaminoglycan able to displace [3H]heparin (IC50, 3.2 x 10(-7) M), although it was 10-fold less effective than heparin itself (IC50, 3.6 x 10(-8) M). Scatchard analysis revealed the existence of high-affinity heparin binding sites (Kd, 5 x 10(-8) M). Furthermore, heparin inhibited serum-induced stimulation of inositol lipid turnover. Taken together, these results indicate that heparin inhibits BC3H-1 cell growth by interacting with the cell surface, possibly disrupting the flow of growth factor-related mitogenic signalling.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Lipolytic activity of whole isolated liver cells in aqueous suspension.

1. Liver contains a lipase which catalyzes in vitro the hydrolysis of esters of short-chain normal primary alcohols and fatty acids. It is shown that this enzymatic activity can be measured by using intact liver cells as source of enzyme. During short-term incubations of suspensions of cells isolated from rat liver, the lipase acts as a membrane-bound enzyme and readily attacks [3H] oleoylethanol added as an emulsion into the bathing medium. The lipolytic reaction proceeds linearly for at least 20 min at 37 degrees C, at the pH optimum of 8.5. [3H] Oleic acid, a reaction product, is mostly retained in the medium and is used to monitor the lipolytic process. 2. In the presence of heparin, the bound lipase is released in the medium in amounts representing one-third to one half the total activity contained in the cells. This release is very rapid and associated in all cases with a concomitant release of lactate dehydrogenase activity. Such effects are consistent with the interpretation that heparin, at concentrations comprised between 10 and 100 mug per ml, causes alterations of the plasma membrane of the isolated cells, resulting in the dispersion of membrane-bound and cytoplasmtic material. This action of heparin is totally blocked by protamine sulfate (1 mg/ml). No specific effect of heparin directed towards the selective release of lipase could be demonstrated under these conditions. 3. During incubations in the presence of heparin, it was observed that the release of monoester lipase was quantitatively related to a simultaneous decrease in membrane-bound as well as in total monoester lipase activity measureable in the cells after homogenization. This, along with the reappearance of membrane-bound activity immediately after heparin withdrawal, suggest that under the experimental conditions, the membrane-bound enzyme is replaced from inside the cell in proportion of its release by heparin.     

Effect of pentosan polysulphate, standard heparin and related compounds on protein kinase C activity.

Ca2+/phospholipid-dependent protein kinase (PKC) was inhibited by sulphated polysaccharides. Pentosan polysulphate (PPS) and heparin were 8-10-times more potent than dextran sulphate or heparan sulphate. Steady-state studies revealed that PPS was a competitive inhibitor with respect to ATP with an apparent Ki value of 0.32 micrograms/ml and a non-competitive inhibitor with respect to histones. In contrast, the inhibition of PKC by heparin was competitive with substrate and non-competitive with respect to ATP. The interaction of sulphated polysaccharides with the catalytic domain of PKC was further demonstrated by the absence of effect on [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC. Furthermore, PPS and heparin inhibited equally cAMP-dependent protein kinase and tyrosine protein kinase. Structure-function relationships indicated that the Inhibition of protein kinases by PPS and heparin fractions was highly dependent on molecular weight. Additionally, PKC-affinity chromatography revealed that a high-molecular-weight heparin fraction with strong anti-PKC activity was eluted. We set out to demonstrate that heparin and PPS, which are potent antiproliferative agents on vascular smooth muscle cells (SMC), alter intracellular PKC activity (both membrane and cytosolic). Therefore, it is suggested that the mechanism by which sulphated polysaccharides inhibit SMC growth may be by direct inhibition of PKC in SMC.     

17β-estradiol inhibits the production of infectious particles of hepatitis C virus

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E₂, but not P₄, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E₂–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E₂, inhibits the production of HCV infectious particles in an ERα–dependent manner.     

Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection

To elucidate the anti-ulcer potential of Cladosiphon fucoidan, anti-peptic activity, bFGF stabilizing activity and inflammatory properties of this and related substances were investigated. Anti-peptic activity was observed with this and other sulfated polysaccharides such as dextran sulfate, carrageenan, and Fucus fucoidan. However, non-sulfated polysaccharides such as mannan and dextran did not exert the anti-peptic activity. The loss of bFGF bioactivity was prevented by all sulfated polysaccharides tested except chondroitin sulfate, at pH 7.4 and at pH 4.0. At pH 2.0, only heparin protected the bFGF activity. The generation of superoxide by macrophages and PMNs was stimulated by dextran sulfate, carrageenan, and Fucus fucoidan, whereas Cladosiphon fucoidan, heparin and chondroitin did not. Dextran sulfate, carrageenan, and Fucus fucoidan also stimulated the secretion of TNFalpha from macrophages, while Cladosiphon fucoidan did not. Thus, Cladosiphon fucoidan is a sulfated polysaccharide without inflammatory action. These results suggest that Cladosiphon fucoidan is a safe substance with potential for gastric protection.     

Effect of cycloheximide, beta-D-xylosides and beta-D-galactosides on heparin biosynthesis in mouse mastocytoma.

Heparin biosynthesis has been investigated with mouse mastocytoma in vitro. Minced tumour tissue catalysed the incorporation of [35S]sulphate and [3H]glucosamine into heparin and to a smaller extent into chondroitin sulphate. Addition of cycloheximide caused an inhibition (greater than 80%) of incorporation of each labelled precursor into both polysaccharides. Addition of benzyl beta-D-xyloside relieved the inhibition of incorporation into chondroitin sulphate and restored it to more than threefold that of the control incubation. The effect of beta-D-xyloside on incorporation into heparin was less marked although a consistent small increase of incorporation into this polysaccharide was observed. beta-D-Xyloside did, however, cause a marked incorporation of 35S and 3H labels into material of low molecular weight, which appeared to comprise heparin-like fragments. It is proposed that these fragments arise through a breakdown of the usual process of heparin biosynthesis.     

多糖抗病毒作用研究进展Ⅲ.卡拉胶及其抗病毒作用

卡拉胶是源于某些红藻的天然硫酸多糖,具有多种生物活性。其显著的抗病毒活性,尤其是抗艾滋病HIV1活性,最近得以进一步证实。本文综述了卡拉胶的抗病毒作用,并展望其临床应用前景。     

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).     

The V3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and CD4-derived synthetic peptides.

gp120 is the envelope glycoprotein found on the surface of human immunodeficiency virus type 1 (HIV-1), and it binds to human cell surface CD4 receptors to initiate the HIV-1 infection process. It is now well-established that synthetic peptides from the V3 region on gp120 elicit antibodies that block HIV-1 infection and HIV-1-mediated cell fusion. Here we show that synthetic peptides derived from similar V3 regions of several isolates of HIV-1 bind [3H]heparin, and we also demonstrate that [3H]heparin binds to recombinant gp120 IIIB. The binding could be blocked by unlabeled heparin, dextran sulfate, and by a highly anionic benzylated synthetic peptide derived from human CD4 (amino acids 81-92). The nonbenzylated peptides from the same region were considerably less active. Unlabeled heparin, dextran sulfate, and the CD4-derived peptides were able to compete with the binding of soluble gp120 to immobilized antibodies against fragments of the V3 from isolate IIIB, but they had no effect on the binding of gp120 to anti-peptide antibodies targeted against another unrelated region of gp120. Biotin conjugated to the benzylated CD4-peptide bound to gp120 and was blocked from this binding by anti-V3 antibodies. These results indicate that the three materials that have been demonstrated by others to block HIV-1 infection in vitro, sulfated polysaccharides, certain CD4-derived synthetic peptides, and anti-V3 antibodies, may be acting through a common mechanism that includes binding to the V3 region of gp120 on HIV-1.     

New oligosaccharides from heparin and heparan sulfate and their use as substrates for heparin-degrading enzymes

Oligosaccharides were isolated from heparin and heparan sulfate by a procedure consisting of three major steps: (a) acid hydrolysis; (b) gel chromatography; and (c) cation exchange chromatography on an amino acid analyzer. To date, six new oligosaccharides have been isolated by this procedure and have been sequenced by a combination of NaB3H4-labeling and deaminative cleavage with nitrous acid. The structures of these oligosaccharides were as follows: 1. GlcN-GlcUA-GlcN 2. GlcN-IdUA-GlcN 3. GlcN-GlcUA-GlcN-GlcUA-GlcN 4. GlcN-IdUA-GlcN-GlcUA-GlcN 5. GlcN-GlcUA-GlcN-IdUA-GlcN 6. GlcN-IdUA-GlcN-IdUA-GlcN The linkage positions and anomeric configurations were assumed to be the same as in the polysaccharides from which the oligosaccharides originated. The usefulness of some of these oligosaccharides as enzyme substrates was tested after appropriate modifications and radioactive labeling. Oligosaccharides 2 and 3 were N-[35S]sulfated and were found to serve as substrates for heparan N-sulfate sulfatase (heparin sulfamidase), with a homogenate of cultured skin fibroblasts as enzyme source. Similarly, reduction of oligosaccharide 2 with NaB3H4 yielded a substrate for acetyl-CoA:alpha-D-glucosaminide N-acetyltransferase. Finally, the previously known disaccharide, 4-O-alpha-D-glucosaminyl-L-iduronic acid, which was isolated in the course of this work, was N-acetylated with [3H] acetic anhydride and was shown to be a substrate for N-acetyl-alpha-D-glucosaminidase.     

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.     

Structural features and in vitro antiviral activities of sulfated polysaccharides from Sphacelaria indica

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance to virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study, we have analyzed xylogalactofucan- and alginic acid-containing fractions from Sphacelaria indica, a marine alga. The xylogalactofucan that has apparent molecular mass of 26±5 kDa and negative specific rotation [α](D)(32) -71° (c 0.2, H(2)O) contains, inter alia, (1→3)-linked L-fucopyranosyl and D-galactopyranosyl residues. The algin (molecular mass: 21±5kDa) contains 41% guluronic and 59% mannuronic acid residues. The 50% inhibitory concentration (IC(50)) values of these macromolecules and their chemically sulfated derivatives against herpes simplex virus type 1 (HSV-1) were in the range of 0.6-10 μg ml(-1) and they lacked cytotoxicity at concentrations up to 200 μg ml(-1). The antiviral activity was dependent on the sulfate contents of the polysaccharides. The results support the feasibility of inhibiting HSV infection by direct interaction of polysaccharides with viral particles.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.