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N Parthasarathy

Publications and source records attributed to N Parthasarathy.

At least 19 recordsLinked to original sources
PubMed1998

Oligosaccharide sequence of human breast cancer cell heparan sulfate with high affinity for laminin.

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) --> AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5[IdoA(2-SO4) --> AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5, [IdoA(2-SO4) --> GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

Amino Acid Sequence↗
PubMed1997

Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone.

A unique feature of lipoprotein lipase (LpL), the rate-limiting enzyme in the hydrolysis of circulating triglycerides, is its movement from its cell of synthesis, adipocyte or myocyte, to its site of action, the luminal endothelial surface. This involves processes that allow LpL to be released from the adipocyte cell surface and transferred against the flow of interstitial fluid to the luminal surface of endothelial cells. LpL, an unstable enzyme, must retain its activity during this process. Whether a chaperone-like molecule is involved in LpL stabilization and transport is unclear. In the present study, we tested the hypothesis that endothelial cells secrete factors that release LpL and promote its transfer to the luminal endothelial surface. Incubation of adipocytes with endothelial cell conditioned medium (ECCM) led to release of about 2-fold more LpL activity than control medium. Medium from endothelial cells exposed to lysophosphatidylcholine (lyso-ECCM), a product of LpL lipolysis of lipoproteins, released approximately 3-fold more LpL than ECCM. Concomitant with the release of LpL, adipocyte cell surface heparan sulfate (HS) proteoglycans were degraded suggesting that lyso-ECCM contained a heparanase-like activity. More heparanase was found in media from the basolateral than the apical side of lysolecithin-stimulated polarized endothelial cells. In coculture experiments, lipolysis and lysolecithin stimulation of endothelial cells increased LpL release from adipocytes. LpL released by lyso-ECCM remained stable and did not lose enzymatic activity at 37 degrees C for 1 h. LpL activity was also stabilized by heparanase-digested fragments of HS (HS oligosaccharide) and by purified LpL binding decasaccharide. Moreover, LpL.HS oligosaccharide complexes crossed endothelial cell monolayers and bound to the apical side of the cells. Thus, an endothelial heparanase may play a critical role in releasing subendothelial HS bound proteins, and specific HS oligosaccharides produced by this enzyme may serve as extracellular chaperones.

Adipocytes↗
PubMed1996

Isolation of heparin-derived oligosaccharides containing 2-O-sulfated hexuronic acids, by lipoprotein lipase affinity chromatography.

Oligosaccharides (hexa to dodeca) terminating with [3H]2,5-anhydromannitol (AManR) were isolated from heparin by partial cleavage with nitrous acid at low pH (pH 1.5) followed by gel filtration and reduction with [3H]NaBH4. They were subsequently chromatographed on a lipoprotein lipase (LpL)-Sepharose column. High- and low-affinity oligosaccharides for LpL were isolated and characterized. Disaccharide analysis revealed the presence of (IdceA(2-SO4)-->AManR6-SO4) and (IdceA(2-SO4)-->AManR) as the major disaccharide products after low pH nitrous acid treatment. The oligosaccharides are, therefore, enriched in IdceA(2-SO4)-(GlcNSO4 +/- 6-SO4) sequences. Furthermore, they are found to be composed of 2-O-sulfated hexuronic acid-containing sequences, structural features, characteristic of heparin and heparan sulfate oligosaccharides with potential antiproliferative activities. These oligosaccharides may have the potential as lipase-releasing agents from endothelial and adipocyte surfaces.

Borohydrides↗
PubMed1995

Specific regulation of procoagulant activity on monocytes. Intrinsic pathway inhibition by chondroitin 4,6-disulfate.

Hypercoagulability of blood, monocytic infiltration, and changes in pericellular and extracellular matrix glycosaminoglycans (GAGs) are observed in atherosclerosis, inflammation, and neoplasia. In the present studies, monocyte procoagulants and different GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and heparan sulfate, were tested either in clotting assays with whole plasma or in chromogenic assays with purified coagulation proteases. Procoagulant activity in plasma was inhibited by three of the seven GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of purified coagulation protease was inhibited only by CSE, and the inhibition was observed with intrinsic (factor VIIIa/IXa) but not extrinsic (tissue factor/factor VII) components. Reciprocal titration experiments with enzyme and substrate and Scatchard type analyses were consistent with concentration-dependent inhibitory interactions between CSE and sites on both factor VIIIa and IXa. On purified phospholipids, CSE concentration resulting in half-maximal inhibition (Ki) was 5 ng/ml for interaction with factor IXa and > 500 ng/ml for interaction with factor VIIIa. The Ki values were lower for reactions on purified lipid than for reactions on monocyte surfaces and for reactions on resting than on endotoxin-stimulated monocytes. Experiments with CSE oligosaccharides of defined size indicated that the smallest CSE fragment capable of inhibitory activity was composed of 12-18 monosaccharide units. Collectively, these results indicate that factor X-activating reactions are inhibited by GAGs expressed on monocyte membranes. Inhibition is specific with respect to the structure of both the GAG and the activating protease. Lack of inhibition by added CSA, CSB, and CSC in contrast to CSE strongly suggests a direct role of 4,6-di-O-sulfated N-acetylgalactosamine GAG structures in the inhibition of intrinsic pathway protease. These findings also suggest potential pharmacologic use of CSE as specific anticoagulant in the management of prothrombotic states mediated by intrinsic pathway coagulation reactions.

Brain↗
PubMed1994

Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.

Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.

Animals↗
PubMed1991

The major proteoglycan of adult rabbit skeletal muscle. Relationship to small proteoglycans of other tissues.

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis. Two major products were obtained when the intact proteoglycans were cleaved by CNBr, and those peptides were separated by SDS/PAGE and by ion-exchange chromatography. Sequencing of the N-terminal amino acids of either the intact proteoglycans or the CNBr-cleaved products allowed for comparison of the muscle and tendon proteoglycan with derived amino acid sequences previously reported for bovine bone proteoglycan. The bone and tendon proteoglycan sequences were remarkably similar, whereas those of the muscle proteoglycan differed from the other two molecules. The major site of glycosaminoglycan substitution was on a peptide fragment distant from the N-terminus, and a presumptive serine residue at position 4 from the N-terminus also appeared to be substituted, perhaps with a small glycosaminoglycan chain. These results provide some insight into the diversity of small proteoglycans of the PG-II class and provide a basis for exploring their mode of genetic expression.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle.

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

Quantitative method for the determination of glycosaminoglycans from small quantities of glomerular basement membranes.

A simple, sensitive, and efficient method is described for the quantitative determination of glycosaminoglycans from bovine renal glomerular basement membranes. After release from glomerular basement membrane by protease treatment, the glycosaminoglycans were isolated by a modified DEAE-cellulose column chromatographic procedure. Quantitation of glycosaminoglycans was achieved by hexuronate measurements with a microadaptation method employing m-hydroxydiphenyl. This procedure has proven useful in analyzing small samples of basement membranes as little as 0.75 mg. The glycosaminoglycan was identified as heparan sulfate by cellulose acetate electrophoresis and nitrous acid treatment.

Animals↗
PubMed1984

Isolation and characterization of the heparan sulfate proteoglycan of the bovine glomerular basement membrane.

A proteoglycan, accounting for about 75% of the total hexuronic acid of the bovine glomerular basement membrane, was solubilized by 4 M guanidine HCl extraction and purified by filtration on Sepharose CL-6B. This glycoconjugate was found to have an apparent molecular weight of 200,000 and to consist of peptide and carbohydrate in a ratio of 70 to 30. The amino acid composition of the proteoglycan was notable for its high content of half-cystine (61/1000 total amino acid residues). Alkaline NaB3H4 treatment of the proteoglycan released heparan sulfate chains terminating in [3H]xylitol with an Mr approximately 14,000 (hexuronic acid/xylitol = 30:1). Four such glycosaminoglycan units were calculated to be present in each proteoglycan molecule, and on the basis of previous studies (Parthasarathy, N., and Spiro, R. G. (1981) J. Biol. Chem. 256, 507-513), these appear to be clustered in a very limited segment of the polypeptide. Nitrous acid degradation of the NaB3H4-reduced chains yielded radiolabeled oligosaccharides derived from the xylitol end; the relatively large size of these fragments (average Mr approximately 6,000) indicated an uneven distribution of N-sulfate with a preferential location of these groups in the peripheral regions of the glycosaminoglycans. The occurrence in the proteoglycan of sugars such as galactosamine, sialic acid, and mannose, which are not constituents of heparan sulfate, suggested that other O-linked as well as N-linked carbohydrates occurred in the molecule. Evidence for small serine (threonine)-bound units was provided by alkaline NaB3H4 treatment of the proteoglycan which converted 85% of the galactosamine to 3H-labeled galactosaminitol present in Bio-Gel P-2 included oligosaccharides (14 mol/mol proteoglycan).

Amino Acids↗
PubMed1984

X-ray diffraction studies on cation-collapsed DNA.

The polyamines spermidine, spermine and putrescine are now known to induce tertiary collapse of DNA. In this collapsed state DNA assumes a compact toroidal conformation. However, the structural details of DNA in these compact particles and the forces that stabilize the collapsed state are not clear. We show here that the structural arrangement of DNA in this tertiary conformation is determined by the chemical structure of the agent used to collapse. We have used aliphatic triamines (NH+3--(CH2)3--NH+2--(CH2)n--NH+3 with n = 3, 4, 5 and 8) and diamines (NH+3--(CH2)x--NH+3 with x = 2, 3, 4 and 6) to collapse DNA. We find that the Bragg spacing and the calculated interhelical spacing for a hexagonal packing model vary systematically with the length of the methylene bridge. We also find that the ionic strength of the solution has no effect on the Bragg spacing. This observation suggests that the arrangement of DNA strands in the complexes is determined by the structure of the polycation, and argues against suggestions that the structure of the collapsed state is maintained by the balance of long-range electrostatic repulsive and attractive forces. Instead we propose that DNA helices form a hexagonal array with counterions in the interstices between the helices resulting in a stable three-dimensional phase with high structural order. Arguments are presented favoring such a model in terms of stabilizing and destabilizing thermodynamic forces.

Animals↗
PubMed1984

Influence of phospholipase C on muscarinic acetylcholine receptor binding in rat brain.

Treatment of neural membranes from rat cerebral cortex with phospholipase C (phosphatidylcholine cholinephosphohydrolase) inhibited the binding of radiolabelled antagonists to muscarinic acetylcholine receptors. This inhibition was incomplete, was not competitive, and did not appear to be related to the production of inhibitory products. The affinity of carbamylcholine for cortex muscarinic receptors was increased by phospholipase C action. The distribution of receptors between states of high and low affinity was not affected by phospholipase C; rather, the affinity for carbamylcholine of the lowest affinity receptors was selectively increased. This suggests that membrane lipids influence the interaction of the receptor binding subunit with other structures in the synaptic membrane.

Animals↗
PubMed1982

Effect of diabetes on the glycosaminoglycan component of the human glomerular basement membrane.

The glycosaminoglycan (heparan sulfate) component of glomerular basement membranes from human kidneys of diabetic and nondiabetic subjects has been quantitated after isolation from protease digests of the membranes on DEAE-cellulose microcolumns. A significant decrease (P less than 0.005) in the glycosaminoglycan content of diabetic membranes was observed. Heparan sulfate was identified as the predominant glycosaminoglycan in both diabetic and control subjects and the extent of its sulfation appeared to be similar. The reduced level of glycosaminoglycan in the diabetic glomerular basement membrane was accompanied by a significant elevation of hexoses, which are primarily associated with the collagen component, suggesting that a redistribution of basement membrane macromolecules occurs in the diabetic state. Since heparan sulfate has been implicated as a major component of the glomerular anionic filtration barrier, its decreased content in diabetic basement membranes may contribute to the proteinuria observed in this disease.

Adult↗
PubMed1978

Hydroxyproline-rich glycoprotein in the seed coat of field bean.

1. The seed coats of field bean (Dolichos lablab var. lignosus) were found to contain bound hydroxyproline (0.26 g per 100 g dry weight) extractable in 30% with 5% trichloroacetic acid or in 90% with 10% potassium hydroxide. 2. The alkaline extract was fractionated by DEAE-cellulose chromatography into several hydroxyproline-containing fractions, composed of protein and carbohydrate in varying proportions. From the major fraction, a hydroxyproline-containing glycoprotein fraction was isolated by Sephadex G-200 chromatography. It gave a single band on agar-gel electrophoresis, and contained: protein (23.1%), arabinose (11.2%), galactose (13.4%), glucose (17.6%), mannose (22.6%) and uronic acids (11.3%). The content of hydroxyproline in the protein moiety was about 9%. Hydrolysis of the glycoprotein fraction with barium hydroxide yielded three components containing hydroxyproline and arabinose at the ratios of 1:2, 1:3 and 1:4.

Chromatography, DEAE-Cellulose↗