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We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the polypeptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophytohemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another noteworthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(O) structures. In the present study, this glycoprotein was shown to contain mono- (0.4% of the total oligosaccharides), bi-(78.2%), tri- (12.3%), and tetraantennary (2.3%) complex type oligosaccharides in addition to a series of high mannose type oligosaccharides, Man6-9GlcNAc2 (0.8%). Biantennary complex type oligosaccharide chains were those with (8.2%) and without (70.0%) a bisecting GlcNAc residue and approximately 13.2%, 2.2%, and 0.4% of these contained blood group H(O), A, and B structures, respectively. The tri- and tetraantennary complex type chains were those with and without N-acetyllactosamine repeats, and about 13.0% of the triantennary chains without the N-acetyllactosamine repeat contained the blood group H(O) structure. Occurrence of these asparagine-linked oligosaccharides with blood group A and B structures suggest that the repeated use of factor VIII/vWF pooled concentrate for the treatment of hemophiliacs could result in the production of antibodies against vWF with a different blood group from that of the patient, and this development may be pathogenic.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.

A previous study identified a Chinese hamster ovary cell mutant, pgsE-606, which is defective in the N-sulfotransferase that catalyzes one of the initial polymer-modification reactions in the biosynthesis of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065). The structure of heparan sulfate generated by these cells reflects a 3-5-fold reduction in enzyme activity. The mutant produces heparan sulfate with half the content of N-sulfated glucosamine residues of that produced by wild-type cells and a more sparse distribution of N-sulfated residues. The present study demonstrates corresponding reductions in the proportion of 6-O-sulfated glucosamine residues (41% reduction) and the content of L-iduronic acid (51% reduction). The amount of 2-O-sulfated L-iduronic acid declines more dramatically (from 25% of total L-iduronic acid in the wild type to 8.4% in the mutant). Enzymatic assay of mixed O-sulfotransferases showed that the mutant has more activity than the wild type. Previous studies on the biosynthesis of heparin/heparan sulfate in cell-free systems point to a pivotal role of N-sulfation in determining the extent of the subsequent polymer-modification reactions. The present study shows that this concept also applies to heparan sulfate biosynthesis in the intact cell.

Previous studies on rabbit thrombomodulin (TM) revealed that certain anticoagulant activities expressed by TM depend on the presence of an acidic domain tentatively identified as a sulfated galactosaminoglycan (Bourin, M.-C., Ohlin, A.-K., Lane, D., Stenflo, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 8044-8052). The glycan was released by alkaline beta-elimination, isolated by ion-exchange chromatography, and radiolabeled by partial N-deacetylation (hydrazinolysis) followed by re-N-[3H]acetylation. The labeled product behaved like standard chondroitin sulfate on ion-exchange chromatography, exhibited a Mr of 10-12 x 10(3) on gel chromatography, and was susceptible to degradation by chondroitinase and testicular hyaluronidase. The major labeled degradation products following digestion of the glycosaminoglycan with chondroitinase were identified, depending on the incubation conditions, either as 4/6-mono-O-sulfated, 4,5-unsaturated disaccharides (delta HexA-GalNAc(S] and N-acetylgalactosamine 4,6-di-O-sulfate (GalcNAc (diS], the latter component accounting for approximately 25% of the total label, or as a major fraction of labeled trisaccharide, with the predominant structure GalNAc(diS)-GlcA-GalNAc(diS). The terminal GalNAc(diS) unit (not substituted at C3) was shown to be more susceptible to N-deacetylation during hydrazinolysis than were the internal GalNAc units (substituted at C3), and thus was more extensively labeled, resulting in over-representation of this unit. It is concluded that rabbit TM is a chondroitin sulfate proteoglycan, which carries a single glycan side chain characterized by an unusual accumulation of sulfate groups at the nonreducing terminus. Metabolically 35S-labeled TM was isolated from cultured rabbit heart endothelial cells and characterized as a chondroitin sulfate proteoglycan which accounted for 1-2% of the total 35S-labeled cell-associated macromolecules. The isolated chondroitin sulfate showed weaker antithrombin-dependent anticoagulant activity, on a molar basis, than the intact TM proteoglycan. The anticoagulant action of TM thus depends on a unique form of functional collaboration between the different constituents of a glycoconjugate.

The N-linked oligosaccharide structures on the envelope glycoprotein gp120 of human immunodeficiency virus 1 derived from chronically infected lymphoblastoid (H9) cells have been investigated by enzymatic microsequencing after release from protein by hydrazinolysis, labeling with NaB3H4, and chromatography on adsorbent columns of Phaseolus vulgaris erythrophytohemagglutinin and Ricinus communis agglutinin (Mr 120,000) and on Bio-Gel P-4. A substantially greater diversity of oligosaccharide structures was detected than among those released by hydrazinolysis from recombinant gp120 produced in Chinese hamster ovary cells and investigated by similar procedures (Mizuochi, T., Spellman, M.W., Larkin, M., Solomon, J., Basa, L.J., and Feizi, T. (1988) Biochem J. 254, 599-603) and among those released by endoglycosidases from virus-derived gp120 isolated from infected H9 cells after metabolic labeling with D-[2-3H]mannose or D-[6-3H]glucosamine (Geyer, H., Holschbach, L., Hunsmann, G., and Schneider, J. (1988) J. Biol. Chem. 263, 11760-11767). In this study, 16% of the oligosaccharides were identified as complex-type bi-, tri-, and tetraantennary sialo-oligosaccharides with bisecting N-acetylglucosamine residues. Such structures were lacking on recombinant gp120 and could not be detected on the metabolically labeled, virus-derived glycoprotein. As in the earlier investigations, complex-type chains lacking bisecting N-acetylglucosamine residues, hybrid-type chains, and a series of high mannose-type structures with 5-9 mannose residues were identified. In addition, an array of complex-type chains having one or more outer chains with beta-galactosyl residues were detected in this study, but with additional substitutions that require further investigation. The number of potential N-glycosylation sites on gp120 is on the order of 20, but the oligosaccharide structures are far more numerous. Thus, the salient conclusion from this and earlier investigations is that alternative structures occur on at least some of the glycosylation sites and that numerous glycosylation variants of this glycoprotein are produced even within a single cell line. Since the glycosylation is the product of host cell glycosyltransferases, an even greater number of glycosylation variants of gp120 are predicted to arise from the heterogeneous cell populations harboring the virus in in vivo infection.

An enzymatic method is described for the analysis of the content of 5-methylcytosine in the DNA of eukaryotic cells. The conventional acid hydrolysis procedure was found to result in variable deamination of 5-methylcytosine into thymine, making the exact quantitation difficult. We applied the enzymatic method for the analysis of a simple eukaryotic viral genome. Viral and cell DNA in Simian Virus 40-infected monkey kidney cells was labeled with [methyl-3H]methionine in vivo and their 5-methylcytosine content was analyzed. The content of 5-methylcytosine per thymine in the total intracellular viral DNA was found to be less than 10(-4) of the host DNA. The implication of this result with regard to eukaryotic gene expression is discussed.

The structures of O-glycosidically linked oligosaccharides present in the heparan sulfate and chondroitin sulfate proteoglycans isolated from the culture medium of a normal (HBL-100) and a malignant (MDA-MB-231) human mammary epithelial cell line have been determined. Both proteoglycan types from the two cell lines contain a series of O-linked oligosaccharides ranging in size from di- to hexasaccharide. Cells were grown in the presence of either [3H]glucosamine or [3H]galactose and Na2 35SO4, and the proteoglycans were isolated as described (Gowda, D. C., Bhavanandan, V. P., and Davidson, E. A. (1986) J. Biol. Chem. 261, 4926-4934). The O-linked oligosaccharides were released from the proteoglycans by alkaline borohydride treatment and purified by a combination of gel filtration and high voltage paper electrophoresis. The structures of two neutral and seven acidic oligosaccharides were established based on sugar composition, the results of periodate oxidation, sequential exoglycosidase treatment, and methylation analysis. Periodate oxidation, taking advantage of tritium label at specific positions of constituent sugars, proved to be a valuable tool in establishing the structure of isomeric components in the mixture. The structures of the oligosaccharides were assigned as follows: (Formula: see text) The oligosaccharide containing both sialic acid and ester sulfate is novel and has not been reported previously.

CDP-diglyceride, an important metabolic intermediate in the biosynthesis of phospholipids, has been isolated for the first time from a mammalian tissue. The isolated material, labeled in incubations of intact rat pineal glands with 32P, [3H]cytidine, or [3H]CTP in the presence of DL-propranolol, was chromatographically identical with authentic CDP-diglyceride and was able to serve as phosphatidyl donor in the enzymatic synthesis of phosphatidylinositol and phosphatidyglycerol. It yielded the expected products upon enzymatic and chemical degradation. No dCDP-diglyceride was detected No radioactive CDP-diglyceride was detected following incubations in the absence of propranolol. Stimulation of CDP-diglyceride labeling from 32P1 occurred at propranolol concentrations between 0.03 and 1.0 mM. Net synthesis of the liponucleotide was shown. At 0.1 mM, propranolol incrased the incorporation of radioactivity into phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. When inositol (10 mM) and propranolol (0.1 mM) were both present, phosphatidylinositol labeling was further increased, wheas stimulation of phosphatidylglycerol and CPD-diglyceride labeling was abolished. Since CDP-diglyceride did not accumulate in the absence of the drug, its availability may normally be the limiting factor in phosphatidylinositol and phosphatidylglycerol biosynthesis. When propranol is present, inositol may become limiting and thus may lead to the observed labeling pattern.

Nonsulfated, partially N-deacetylated, 14C-labeled polysaccharide was formed by incubation of a mouse mastocytoma microsomal fraction with UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine. After 60 min at 37 degrees the incorporation of radioactivity was interrupted, and sulfation was initiated, by the addition of unlabeled UDP-glucuronic acid and 3-phosphoadenylylsulfate, respectively. After varying periods of incubation with the nucleotide sulfate, polysaccharide was isolated and analyzed by chromatography on DEAE-cellulose. During incubation with 3-phosphoadenylylsulfate, sulfated 14C-polysaccharides were formed, while the nonsulfated 14C-polysaccharide was eliminated, each type of component retaining the same elution position throughout the entire sulfation period (60 min). No accumulation of low sulfated material was observed. Fully sulfated heparin was detected after only 0.5 min of sulfation. These results suggest that the sulfation of heparin is a rapid process, involving a limited number of polysaccharide molecules at a time. Besides heparin-like products, having both N- and O-sulfate groups, and [14C]iduronic acid as predominant uronic acid constituent, an additional sulfated component was detected, having N-sulfate but no O-sulfate groups. This novel polysaccharide contained [14C]glucuronic acid but little or no [14C]iduronic acid. Previous studies have shown that L-iduronic acid residues in heparin are formed by C-5 inversion of D-glucuronic acid units, previously incorporated into the polymer; the inversion reaction requires concomitant sulfation of the polymer. The present results demonstrate that the formation of iduronic acid residues is related to the sulfation of hydroxyl groups. Pulse-chase experiments with 3-phosphoadenylyl[35S]sulfate indicated that the N-sulfated polymer, lacking O-sulfate groups, could be converted into heparin-like products by further sulfation of hydroxyl groups only. The biosynthesis of heparin may thus involve a pathway, in which the formation of N-sulfated intermediates precedes the introduction of O-sulfate groups.

The fraction of hen egg albumin glycopeptides mixture, which passes through a Dowex 50-H+ column, contains two sulfate-containing glycopeptides. Based on the structural studies of oligosaccharides released from the glycopeptides by hydrazinolysis, their structures were elucidated as follows. (formula; see text)

Exponentially growing cultures of Lactobacillus acidophilus strain 60AM Gasser were previously shown to lose about one-third of their cell wall peptidoglycan per generation via turnover (Boothby, D., Daneo-Moore, L., Higgins, M. L., Coyette, J., and Shockman, G. D. (1973) J. Biol. Chem. 248, 2161-2169). We now show that 20 to 30% of the [3H]lysine initially present in insoluble peptidoglycan fractions was retained after 4 or more generations of continued exponential growth of cultures in the absence of label. Treatment of peptidoglycan fractions, before and after 6 or 8 generations of chase with lysozyme (EC 3.2.1.17), released soluble products containing [3H]lysine which had electrophoretic mobilities identical with the disaccharide-peptide derivatives obtained from the wall peptidoglycan of this species. Because protein is known to contaminate peptidoglycan residues, the double labeled technique was used to show that one-half or less of the label lysine present after 6 or 8 generations of chase could be attributed to protein contamination. This then left a minimum fraction of 10 to 20% of the peptidoglycan that was immune to turnover. The absence of turnover of peptidoglycan labeled during short pulses has now been quantitated to show that pulses shorter than 12% of a generation (6 to 7 min) did not turn over. This turnover-immune fraction is in reasonably good agreement with the immune fraction of 10 to 20% observed after long periods of chase of extensively labeled peptidoglycan.

Epidermal growth factor (EGF)-enhanced protein kinase activity in plasma membrane preparations of A-431 human epidermoid carcinoma cells was shown to involve the phosphorylation of tyrosine residues. Phosphotyrosine was detected in both endogenous membrane proteins and in histone when added as an exogenous protein substrate. The major phosphorylated amino acid in partial acid hydrolysates of 32P-labeled A-431 membranes was identified as phosphotyrosine on the basis of its identical behavior to authentic phosphotyrosine on paper electrophoresis and thin layer chromatography; its 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivative was indistinguishable from that of the authentic compound. Only traces, if any, of phosphoserine or phosphothreonine were detected. [32P]Phosphotyrosine was also detected in pronase digests of 32P-labeled membrane proteins. The EGF receptor . protein kinase complex, which was solubilized with Triton X-100 and purified by EGF affinity chromatography, was shown to phosphorylate tyrosine residues of the isolated membrane protein.

The carbohydrate moieties of gamma-glutamyltranspeptidase purified from rat kidney were released as oligosaccharides by hydrazinolysis. Fractionation of the oligosaccharide mixture by paper electrophoresis and Bi-Gel P-4 column chromatography and structural study of each component by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation have revealed that it is composed of 23 neutral oligosaccharides, monosialyl derivatives of 67 oligosaccharides, disialyl derivatives of 62 oligosaccharides, and trisialyl derivatives of 5 oligosaccharides. The neutral oligosaccharides are either high mannose type or biantennary complex type, and the acidic oligosaccharides are bi-, tri-, and tetranntennary complex type sugar chains. Most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the beta-mannosyl residue of their trimannosyl core. Another characteristic feature of these complex type sugar chains is that they are enriched with nonreducing terminal beta-N-acetylglucosamine residues.

The substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase from a mouse mastocytoma was examined to determine the effects of N-acetyl and O-sulfate groups on substrate recognition by the enzyme. [5-3H]Glucuronosyl-labeled heparosan N-sulfate was prepared enzymatically and was modified chemically by partial N-desulfation and N-acetylation. After enzymatic release of tritium, the location of remaining label was determined by deaminative cleavage and analysis of resulting di-, tetra-, and higher oligosaccharides. This analysis indicated that a D-glucuronosyl residue is recognized as a substrate if it is linked at C-1 to an N-acetylated glucosamine residue and at C-4 to an N-sulfated unit. However, the reverse structure, in which the D-glucuronosyl moiety is bound at C-1 to an N-sulfated residue and at C-4 to N-acetylated glucosamine, is not a substrate. Similar studies with O-sulfated heparin intermediates showed that O-sulfate groups either at C-2 of the L-iduronosyl moieties or at C-6 of vicinal D-glucosaminyl moieties prevent 5-epimerization. These findings were confirmed by studies of the reverse reaction, in which tritium was incorporated from 3H2O into partially O-desulfated heparin and the location of incorporated radioactivity was determined. These and more direct experiments corroborated the previous conclusion that the L-iduronosyl moieties are formed after N-sulfation but before O-sulfation. Assessment of the influence of substrate size on the reaction further showed that a large substrate is preferred; an octasaccharide released tritium at a rate approximately 10% of that observed for the parent polysaccharide, and some release occurred also with smaller oligosaccharides.

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.

Primary structural studies have been carried out on the light chain of a homogeneous rabbit antibody to Group C streptococcal carbohydrate. Approximately 20 g of this antibody were obtained by multiple exchange transfusions at the peak of the antibody response. The isolated antibody was homogeneous by several antigenic and chemical criteria. The light chain was isolated and modified, and then digested with alpha-chymotrypsin or thermolysin. The resulting peptides were isolated by gel filtration, paper electrophoresis, and paper chromatography. The amino acid sequences of these peptides were determined by Edman degradation plus dansylation. This supplied sufficient information to assign approximately 90 percent of the residues in the chain. The destruction of tyrosine during acid hydrolysis of peptides which had been eluted from a paper chromatogram was investigated. This destruction is due to inpurities in the paper which contaminate the peptides. Prevention of such destruction can be achieved by predevelopment of the paper with 1 N NH4OH prior to paper chromatography.

A complete amino acid sequence containing 210 residues of a rabbit light chain of a homogeneous Group C streptococcal antibody has been determined as: See journal for formula. This light chain is allotype b4 and is of the V-K1 subgroup.

A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.

Peptide analogs of angiotensin were phosphorylated in vitro by the src gene product, pp60src, of Rous sarcoma virus. The Km for the phosphorylation reaction varied from 1 to 5 mM and the Vmax varied from 2 to 10 nmol/min/mg. Tyrosine was the only residue phosphorylated in all analogs that were examined. The peptides were phosphorylated by tyrosyl protein kinases associated with several avian sarcoma viruses and by the epidermal growth factor-receptor kinase of A431 cells. Peptide substrate also was used to investigate the effectiveness of three different phosphatase inhibitors. Assay of tyrosyl kinase activities in whole cell lysates indicated that both p-nitrophenyl phosphate and sodium vanadate were potent inhibitors of phosphotyrosine phosphatases.

Heparin was cleaved with nitrous acid at pH 1.5 and the products were reduced with Na+ boro[3H]hydride to generate a mixture of di- and tetrasaccharides having anhydro-D-[3H]mannitol (AManR) residues on their reducing terminals. The products were purified to homogeneity by gel filtration and high-performance liquid chromatography. For each oligosaccharide, the proportions of D-glucuronic acid (GlcUA), L-iduronic acid (IdoUA), N-acetyl-D-glucosamine (GlcNAc), and AManR and the monosaccharide sequence were determined by quantification of the products of acid hydrolysis. The tetrasaccharide sequences were determined by comparison of the disaccharide units formed by hydrazinolysis and deamination with previously characterized disaccharides. The following new oligosaccharides were identified: GlcUA(2-SO4)-AManR, GlcUA(2-SO4)-AManR(6-SO4), GlcUA-AManR(3,6-diSO4), GlcUA-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc-GlcUA-AManR, GlcUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3,6-diSO4), and IdoUA(2-SO4)-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4). Then the disaccharides and the tetrasaccharides were readily resolved by high-performance anion-exchange liquid chromatography and were quantified on the basis of the amount of 3H counts/min in each. The structures are discussed in terms of their implications regarding heparin biosynthesis and anticoagulant activity.

Structural changes in the asparagine-linked sugar chains of plasma membrane glycoproteins during myeloid and monocytoid differentiation were investigated by the use of an in vitro differentiation system for human promyelocytic leukemic cells (HL-60), which can be induced to more mature myeloid cells by exposure to dimethyl sulfoxide and to macrophage-like cells by a phorbol ester. The asparagine-linked sugar chains released from their plasma membranes by hydrazinolysis were separated into a neutral fraction and an acidic fraction composed of over ten components. The content of neutral oligosaccharides, which accounted for 8% of the total asparagine-linked sugar chains in HL-60 cells, increased slightly to 13% in dimethyl sulfoxide-induced cells and markedly to 33% in phorbol ester-induced cells. Structural analyses revealed that the neutral oligosaccharides of HL-60 cells are all of the complex type with a variety of Gal beta 1----4GlcNAc beta 1----units in their outer chain moieties and the following core structure: (sequence; see text) After myeloid and monocytoid differentiation, the total amount of neutral complex-type sugar chains did not change significantly, but newly found high mannose-type sugar chains contributed up to 3% and 24% of the total sugar chains, respectively.

An RNA species from Escherichia coli infected with phage lambda was purified by hybridization to lambda 1-strand DNA and shown to have variable length. This variability was due to a variable number of adenylate residues attached to the 3' end of the molecule. Pancreatic RNase treatment of the RNA-DNA hybrid removed the 3'-terminal adenylate residues, generating a homogeneous RNA molecule terminating with -Up. The results indicate the presence of adenylate residues not coded by the DNA template at the terminus of this transcript from intact cells.