Cell surface lipids and adhesion. IV. The effects of trypsin on lipid turnover by the plasmalemma

1979 ◽  
Vol 38 (1) ◽  
pp. 283-292
Author(s):  
A.S. Curtis ◽  
O. Hill
Keyword(s):  
Free Energy ◽  
Enzyme Treatment ◽  
Embryonic Chick ◽  
Trypsin Treatment ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Coa Ligase ◽  
Surface Lipids ◽  
Lipid Turnover ◽  
Phospholipase A2 Activity

Trypsin treatment of intact cells or isolated plasmalemmae from embryonic chick neural retinae leads to an accumulation of lysophospholipids in the plasmalemmae. Trypsin was used at activities commonly used in cell disaggregation techniques. This accumulation appears to result from the decrease in acyltransferase activity in the plasmalemma produced by enzyme treatment. Plasmalemmal CoA ligase activity is not affected by trypsin treatment. Trypsinization has little effect on plasmalemmal phospholipase A2 activity. These results are discussed in relation to (a) the effects of trypsinization on cell adhesion, and (b) the theory that cells cannot adhere to lecithins because of their fluidity or surface-free-energy values. We propose that the effects of trypsinization on adhesion may in large part be due to the effects on other plasmalemmal proteins. Similarly the inability of cells to adhere to lecithin substrates is simply explained as being due to the lysolecithin that contacting cells release from these substrates.

scholarly journals Modulation of mannose receptor activity by proteolysis

1990 ◽  
Vol 270 (3) ◽  
pp. 771-776 ◽  
Author(s):  
V L Shepherd ◽  
R Abdolrasulnia ◽  
J Stephenson ◽  
C Crenshaw
Keyword(s):  
Cell Surface ◽  
Mannose Receptor ◽  
Receptor Activity ◽  
Trypsin Treatment ◽  
Extracellular Proteases ◽  
Down Regulation ◽  
Intact Cells ◽  
Extracellular Milieu ◽  
Coomassie Blue ◽  
Surface Labelling

Macrophages express a receptor on the cell surface that functions to clear glycoproteins from the extracellular milieu. The activity of this receptor is sensitive to treatment with trypsin. In inflammatory situations, macrophages are activated and exposed to increased levels of extracellular proteases. Under these conditions, mannose receptor activity on the macrophages is diminished. We therefore decided to study the effects of trypsin treatment on the structure and activity of cell-associated and purified receptor that might contribute to the activation-associated receptor down-regulation. Trypsin treatment (1 microgram/ml for 3 h) resulted in the production of a 140 kDa, trypsin-resistant fragment from both intact cells and isolated receptor. This fragment was no longer able to bind ligand. The remaining 35 kDa fragment apparently is further degraded into smaller fragments, since no evidence of this domain was found on Coomassie Blue-stained gels. The 140 kDa fragment retained immunoreactivity and contained at least a portion of the iodinated tyrosine residues following surface labelling with Na125I. Neither calcium nor ligand protected the receptor from proteolysis. In addition, prior treatment with oxidants did not increase the susceptibility of the receptor to trypsin digestion. We conclude from these results that the macrophage mannose receptor is clipped by the serine protease trypsin at the cell surface, resulting in the release and further degradation of the binding domain, and the production of a membrane-associated 140 kDa fragment. This trypsin-mediated down-regulation of receptor activity might be important in controlling glycoprotein clearance during inflammation.

scholarly journals Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry in Dictyostelium discoideum

1998 ◽  
Vol 332 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Ralph SCHALOSKE ◽  
Jürgen SONNEMANN ◽  
Dieter MALCHOW ◽  
Christina SCHLATTERER
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Dictyostelium Discoideum ◽  
Knockout Mutant ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Inhibition Studies ◽  
Higher Doses ◽  
Phospholipase A2 Activity ◽  
Entry Mechanism

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233–238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura 2–dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

scholarly journals Antibodies to the retina N-acetylgalactosaminylphosphotransferase modulate N-cadherin-mediated adhesion and uncouple the N-cadherin transferase complex from the actin-containing cytoskeleton.

1991 ◽  
Vol 113 (2) ◽  
pp. 429-436 ◽  
Author(s):  
J Balsamo ◽  
R Thiboldeaux ◽  
N Swaminathan ◽  
J Lilien
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cell Monolayer ◽  
Neural Retina ◽  
Embryonic Chick ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Dependent Cell ◽  
A Cell

Embryonic chick neural retina cells have at their surface an N-Acetylgalactosaminylphosphotransferase (GalNAcPTase) which is associated with, and glycosylates, the calcium-dependent cell-cell adhesion molecule, N-cadherin (Balsamo, J., and J. Lilien. 1990. J. Biol. Chem. 265:2923-2928). In this manuscript, we demonstrate that antibodies directed against the GalNAcPTase, as well as anti-N-cadherin antibodies, are able to inhibit adhesion of chick neural retina cells to a cell monolayer, to immobilized N-cadherin, or to immobilized anti-N-cadherin antibody. These results indicate that anti-GalNAcPTase antibodies modulate the function of N-cadherin, interfering with the formation of N-cadherin-mediated adhesions. We also demonstrate that actin is associated with the N-cadherin/GalNAcPTase complex and that binding of anti-GalNAcPTase antibodies to intact cells results in dissociation of actin from the complex. We suggest that the GalNAcPTase modulates N-cadherin function by altering its interaction with the cytoskeleton.

scholarly journals THE MAMMALIAN CELL-VIRUS RELATIONSHIP

1959 ◽  
Vol 109 (5) ◽  
pp. 487-504 ◽  
Author(s):  
John J. Holland ◽  
Leroy C. McLaren
Keyword(s):  
Hela Cells ◽  
Salt Concentration ◽  
Temperature Sensitive ◽  
Infective Virus ◽  
Cell Nuclei ◽  
Trypsin Treatment ◽  
Intact Cells ◽  
Virus Adsorption ◽  
Method Of Measurement

Phases of attachment, reception or penetration, and eclipse of Type 1 poliovirus infecting HeLa cells in monolayer were studied. Firm attachment was not completely dependent on salt concentration, and was sensitive to temperature change. Like attachment, progressive resistance of adsorbed virus to inactivation by externally applied antibody was temperature-sensitive. Penetration was shown to be independent of physiologic integrity of cells. Virus in process of penetration was not affected by ribonuclease. Eclipse of adsorbed virus was not dependent on metabolic activity or physical integrity of HeLa cells. Debris from poliovirus-susceptible cells inactivated the virus in a manner similar to the kinetic course of virus adsorption by intact cells, and released cell-associated infective virus in similar amounts. All cells insusceptible to poliovirus infection failed to yield active debris. Virus inactivation by debris, like virus reception by intact cells, was temperature-sensitive. Debris could not inactivate virus adsorbed to cells, or alter the progressive incapacity of antibody to neutralize penetrating virus. The active debris factor was insoluble, was not associated with cell nuclei, was inactivated by fat solvents and trypsin treatment, and was destroyed by beat inactivation or sonic disruption. Anti-HeLa serum applied to cells before exposure to virus reduced the rate of virus adsorption, while antiserum treatment immediately following virus adsorption was ineffective. These findings suggested that the capacity of HeLa and other susceptible cells to adsorb, receive, and eclipse poliovirus was associated with organized cytoplasmic lipoprotein structures not possessed by insusceptible cells. The reaction of virus with receptor substance contained in debris was not readily reversed by treatment shown not to affect virus and to destroy activity of uncombined debris. Sensitivity of poliovirus adsorption by HeLa cells to change in environmental salt concentration or temperature was dependent on the method of measurement.

scholarly journals A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities

1983 ◽  
Vol 214 (1) ◽  
pp. 247-255 ◽  
Author(s):  
M H Rider ◽  
E D Saggerson
Keyword(s):  
Trypsin Treatment ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Isolated Mitochondria ◽  
Low Concentrations ◽  
Equivalent Quantity ◽  
Sensitive Factor ◽  
Labile N ◽  
Z Protein ◽  
Coa Hydrolase

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

1990 ◽  
Vol 68 (4) ◽  
pp. 531-534 ◽  
Author(s):  
Gordon M. Wahler ◽  
Nancy J. Rusch ◽  
Nicholas Sperelakis
Keyword(s):  
Cyclic Amp ◽  
Calcium Channel ◽  
Cyclic Gmp ◽  
Drug Exposure ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Intact Cells ◽  
Channel Currents

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (−68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.Key words: cyclic GMP, calcium channels, calcium current, heart.

scholarly journals Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts

1988 ◽  
Vol 250 (3) ◽  
pp. 653-658 ◽  
Author(s):  
J P Slotte ◽  
E L Bierman
Keyword(s):  
Plasma Membrane ◽  
Cholesteryl Ester ◽  
Time Course ◽  
Plasma Membranes ◽  
Mass Movement ◽  
Extracellular Medium ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Cultured Fibroblasts ◽  
Time Course Study

This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.

Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture

1979 ◽  
Vol 57 (6) ◽  
pp. 573-577 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack ◽  
Dominic K. Yip
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Culture Conditions ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Morphological Maturation ◽  
Adipocyte Precursor

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.

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