Subal Bishayee

Isolation and characterisation of a sialic acid-binding lectin (carcinoscorpin) from indian horseshoe crab Carcinoscorpius rotunda cauda☆

A sialic acid-binding lectin, carcinoscorpin, has been purified to apparent homogeneity in 40% yield from the Indian horseshoe carb, Carcinoscorpius rotunda cauda. This glycoprotein lectin of molecular weight 420,000 was composed of two non-identical subunits of molecular weights 27,000 and 28,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The hemagglutination activity of the lectin was susceptible to guanidine-HCl; modification of tyrosyl and tryptophanyl residues also inhibited the activity although alkylation of the -SH group, reduction of disulfide bonds or modification of amino and carboxyl groups were without any effect. The monomeric form of the lectin produced by succinylation of native protein was inactive in binding to sialoglycoconjugates.

Interaction between Concanavalin A and brain lysosomal acid hydrolases

Abstract All the five sheep brain lysosomal acid hydrolases tested, namely arylsulphatase A, acid phosphatase, β-N- acetylhexosaminidase , β-galactosidase and β-glucuronidase bind with Concanavalin A and form enzymatically active precipitate. This enzyme-Concanavalin A complex can be dissociated by α-methyl- d -glucoside and the dissociation is pH dependent; except for arylsulphatase A which dissociates maximally at pH 9.0, all other enzymes dissociate maximally at pH 4.0. The enzyme-Concanavalin A complex formation is inhibited by α-methyl- d -glucoside. Using the differential dissociation of the enzyme-Concanavalin A complex, these enzymes have been purified to the extent of 30–180-fold over the soluble lysosomal fraction. The dissociation of the enzyme-Concanavalin A complex and the inhibition of its formation by α-methyl- d -glucoside suggest the glycoprotein nature of the enzymes.

Further characterization of the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, named carcinoscorpin, has been isolated from the horseshoe crab Carcinoscorpius rotunda cauda. It is a glycoprotein of molecular-weight 420,000, having two subunits of molecular weight 27,000 and 28,000, both subunits responding to glycoprotein stain. Leucine was detected as the only NH2-terminal amino acid. The sedimentation constant of the native lectin was found to be 12.7 s. On digestion with trypsin, the lectin gave 18 soluble tryptic peptides. This lectin was found to be antigenically unrelated to another sialic acid-binding lectin, limulin, isolated from the horseshoe crab Limulus polyphemus. A lectin-specific disaccharide alcohol namely O-(N-acetylneuraminyl) (2 → 6)2-acetamido-2-deoxy-d-galactitol was found to quench the typical tryptophan fluorescence of the native lectin at 332 nm. The association constant for this interaction was determined spectrofluorimetrically and found to be 1.82 × 103m−1.

Effect of methylamine on internalization, processing and biological activation of epidermal growth factor receptor

Epidermal growth factor (EGF), a mitogenic polypeptide of Mr 6045 interacts with responsive cells through high affinity surface receptors [ 11. The membrane receptor for EGF is a polypeptide of &fr -184 000 [2,3], and interaction of EGF with this surface receptor has been shown to lead to the intracellular generation of an activator of DNA replication [4]. As with many other polypeptide ligands, binding of EGF to the surface receptor has been shown to be followed by endocytic uptake of the hormonereceptor complex leading to subsequent proteolytic degradation of both EGF and receptor within lysosomes [3,5,6]. Our earlier studies had indicated that the phenomenon of EGF-induced receptor internalization may play an essential role in the induction of the message necessary for the mitogenic response [3,4]. We are therefore interested in using various inhibitory compounds which block one or more of the steps in the cascade of cellular reactions that follow hormone-receptor binding, and in studying their effect on the induction of the mitogenic message. There have been reports on the inhibitory effects of various amines on the biological fate of hormones bound to target cells [5,7,8]. Here we report the effect of methylamine (MA) on the interaction of EGF with murine 3T3 cells. We find that the crucial event in hormone-cell interaction which is blocked by MA is not the initial endocytic event, but rather the later intracellular proteolytic processing of hormone and receptor. We also report some of the effects of MA on EGF-induced stimulatory responses,

Fractionation of sialoglycoproteins on an immobilized sialic acid-binding lectin

Abstract Carcinoscorpin, the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda, has been immobilized using Sepharose. The immobilized lectin is shown to resolve the isoenzymes of alkaline phosphatase from sheep brain based on the difference in their sialic acid contents. The noninic detergent Triton X-100 does not interfere with the binding efficiency of the column up to a concentration of 1% when tested with 125I-labeled fetuin. However, the use of hexadecyltrimethylammonium bromide produced a 45% inhibition of binding of the labeled fetuin. The efficiency of various saccharides to elute the bound fetuin from the matrix was determined. O-(N-Acetylneuraminyl) (2 → 6) 2-acetamido-2-deoxygalactitol was shown to be the most powerful agent in competing with the fetuin-lectin interaction. Moreover, d -glucuronic acid was also found to elute the bound fetuin from the immobilized lectin. 125I-Labeled fetuin in which an artificial heterogeneity is created by partial desialylation with neuraminidase resolved into three peaks using the immobilized lectin and a gradient of the disaccharide. It is suggested that this immobilized lectin could be used in the purification and resolution of minute amounts of several sialoglycoproteins.

Insulin degradation in 3T3-L1 adipocytes: role of the endocytic lysosomal pathway.

Abstract Insulin bound to 3T3-L1 adipocytes at 12 °C rapidly becomes processed to higher and lower molecular weight components at 37 °C. A part of this insulin processing (degradation) appears to have no role in the expression of its biological effects on hexose and amino acid transport. Degradation is strongly (~70%) inhibited by bacitracin, and very weakly inhibited (~5%) by methylamine, monodansylcadavarine, and bromophenacylbromide. All the other compounds tested for inhibition—azide, dinitrophenol (inhibitors of energy-dependent endocytosis), chloroquine (a lysosomotropic agent), chlorpromazine, phenylglyoxal (reported inhibitors of macromolecular internalization)—inhibited degradation partially to about the same extent (~20%), suggesting that the endocytic lysosomal pathway accounts for only a fifth of insulin degradation in 3T3-L1 adipocytes.

Aberrant energy metabolism in a variant epidermal growth factor receptor-negative fibroblastic cell line.

A variant 3T3 cell line has been isolated [l] lacking mitogenic response to epidermal growth factor [2,3]. This non-responder variant (NR-6) lacked epidermal growth factor (EGF) receptors, but retained the ability to respond to other mitogens, and displayed growth control in culture [ 11. NR-6 cells have been used as specificity controls in identification of the EGF receptor [4-61 on 3T3 cells and for studies on insertion of exogenous hormone receptors into receptornegative mutant cells [7]. We were interested in further character~ing this variant cell line, and examined whether the lack of EGF receptors is accompanied by other losses as well. Such studies may provide new insights into receptor genetics and the regulatory mechanisms involved in receptor biosynthesis. Here we report our preliminary findings on the energy metabo~sm characteristics of NR-6 cells. We find that compared with the parent 3T3 cells, the variant NR-6 cells are extremely deficient in cytochrome c oxidase, a key enzyme in the oxidative phosphorylation pathway, but this deficiency is compensated for by an exceptionally high rate of aerobic glycolysis. This aberrant energy metabolism does not appear to be associated with any transformed growth characteristics.

Differential localisation of phosphorylated and non-phosphorylated forms of arylsulfatase A in lysosomes

The phosphohexosyl components of the lysosomal acid hydrolases are believed to form a part of the common recognition marker for the receptor mediated uptake of these glycoproteins by fibroblasts [l-5]. Depending upon the rate of uptake of a particular enzyme by cultured cells, a ‘high uptake’ form may be distinguished from a ‘low uptake’ form of the same enzyme in the same system [6]. The ‘high uptake’ form of /3-glucuronidase [7], which is efficiently and specifically pinocytosed by fibroblasts, is more acidic than the ‘low uptake’ or poorly pinocytosed form; alkaline phosphatase treatment of the former enzyme abolishes its ‘high uptake’ behaviour without diminishing its catalytic activity. Most lysosomal hydrolases are known to exist in two states, the membrane-bound state and the free (non-latent) state [8]. The physiological significance of these two states is poorly understood. Here we show that the phosphorylated high uptake form of arylsulfatase A (arylsulfate sulfohydrolase EC 3.1.6.1) from sheep brain is a membrane-bound enzyme whereas the free non-phosphorylated ‘low uptake’ form is a soluble protein. This is perhaps the first report demonstrating that the high and low uptake forms of a lysosomal enzyme may be correlated with the physiological microenvironments in which these enzymes exist.

A hepatic membrane-associated factor stimulates nuclear DNA synthesis in cultured fibroblastic cells

Abstract Nuclear DNA replication in cultured mouse fibroblasts is stimulated by isolated hepatic plasma membranes in a time- and concentration-dependent manner. The plasmalemmal activity is susceptible to trypsin treatment, and to treatment with protein modifying agents, N-ethylmaleimide, N-bromosuccinimide, and 2-hydroxy-5-nitro-benzylbromide.

[49] Spontaneous transfer of exogenous epidermal growth factor receptors into receptor-negative mutant cells

Publisher Summary This chapter focuses on the spontaneous transfer of exogenous epidermal growth factor (EGF) receptors into receptor-negative mutant cells. Receptor-EGF interaction produces a variety of rapid events, including activation of a receptor-associated protein and receptor clustering and endocytosis. One or more of these events leads to the intracellular generation of macromolecular activator(s) of DNA replication. The mechanism of receptor action is examined with a variant cell line NR-6 derived from mouse 3T3 that can neither bind nor biologically respond to EGF. Development of a procedure for receptor transfer is an important step toward elucidation of the mechanism of receptor action and understanding the mode of integration of receptor proteins in the membrane. The chapter describes some of the properties of transfer process. EGF receptor is transferred from donor receptor-enriched membranes to recipient receptorless cells in the absence of added fusogenic agents. The cells are assayed for transferred receptor activity by 125 I-labeled EGF binding, or by measuring EGF-induced stimulation of [ 3 H]thymidine incorporation into DNA.