A novel phosphorylcholine-binding protein from rat serum and its effect on heparin-lipoprotein complex formation in the presence of calcium

An adsorbent was synthesized by attaching 4-aminophenylphosphorylcholine to cyanogen bromide-activated Sepharose. A phosphorylcholine (P-choline)-binding protein from rat serum was adsorbed on this affinity column which was eluted by 4 mM P-choline. The protein separated into two bands of Mr = 47,000 and 24,000 on sodium dodecyl sulfate-polyacrylamide gradient gels and contained 18% carbohydrate. A serum protein factor, precipitable by 30-50% (NH4)2SO4, was previously shown to inhibit Ca2+-heparin-rat serum very low density lipoprotein (VLDL) precipitation reaction, whereas P-choline counteracted the action of this protein (Mookerjea, S. (1978) Can. J. Biochem. 56, 746-752). It is now demonstrated that purified P-choline-binding protein prevents Ca2+-heparin-chylomicron or VLDL complex formation and P-choline reverses the effect of this protein. Antibody to P-choline-binding protein raised in rabbits produced a single precipitin line against the pure antigen. The antiserum, however, did not react against rat serum chylomicron, VLDL, low density lipoproteins, or high density lipoprotein. Human serum appears to lack P-choline-binding protein, since (a) the affinity column did not adsorb any such protein, (b) P-choline had no effect on the Ca2+-heparin-serum lipoprotein precipitation reaction, and (c) an immunodiffusion test against the antiserum was negative. However, when P-choline-binding protein was added to human serum, the lipoprotein precipitation was inhibited, and P-choline counteracted the effect of this protein. Preliminary experiments suggested a stoichiometric interaction between P-choline-binding protein and VLDL. Hydrophilic P-choline groups exposed on the surface of VLDL may possibly interact with the P-choline-binding protein and thereby affect the precipitation of lipoproteins by heparin and Ca2+.     

The role of the female Syrian hamster protein on the interaction between serum lipoproteins and heparin

We have previously shown the effect of phosphorylcholine-binding proteins from rat (PCBP) and rabbit (CRP) on the precipitation of serum lipoproteins by heparin in presence of Ca2+. The present paper describes the effect of a phosphorylcholine-binding protein from the female Syrian hamster (FP) on the lipoprotein precipitation reaction. The precipitation of lipoproteins by heparin was lower in assays using female hamster serum in which FP is a prominent protein, compared with assays with male serum in which FP is present in very low concentration. Depletion of FP from female serum resulted in increased lipoprotein precipitation. The addition of purified FP to assays using human very low density lipoprotein (VLDL) inhibited the precipitation reaction. The precipitation of lipoproteins was also examined using serum from male hamsters treated with diethylstilbestrol and female hamsters treated with testosterone, treatments known to modulate the levels of FP. Results indicate an inverse relationship between serum FP levels from normal and hormone-treated hamsters and the precipitation of lipoproteins from their serum. The partially desialylated FP when added to precipitation assays using human VLDL resulted in reduced inhibition of VLDL precipitation.     

Phosphorylcholine: a specific promoter of reparin and serum -lipoprotein interaction

The mechanism of formation of heparin-β-lipoprotein complex was studied. Precipitation of fatty acid labelled β-lipoproteins and optical density changes of the reaction were used as measures for the formation of insoluble complex. Equimolar amounts of Ca-phosphorylcholine were found to be more effective than CaCl₂ to form the insoluble complex. In presence of 10 mM CaCl₂, minute amounts (2–16 μM) of Ca or Na-phosphorylcholine increased the interaction between heparin and serum β-lipoprotein. Equimolar amounts of choline, ethanolamine, acetylcholine, phosphoserine, phosphothreonine, α-glycerophosphate, α-glycerophosphate plus choline, NaH₂PO₄, galactose, glucosamine and N-acetylglucosamine had no effect on the reaction. The results suggested that phosphorylated quaternary nitrogen groups play a role in the interaction of β-lipoprotein with heparin and divalent cation.     

人血清脂蛋白与糖胺聚糖及人主动脉蛋白聚糖的相互作用

本文报道了在[Ca~(2+)]=30mmol/L时,人血清或人血清脂蛋白与各种糖胺聚糖(GAG)及人主动脉两种蛋白聚糖(PG)的相互作用。GAG与血清的作用能力为6—硫酸软骨素(C6—S)>肝素(Hep)>4—硫酸软骨素(C4—S)>透明质酸(HA)>硫酸皮肤素(DS)。极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)可与肝素作用形成不溶性复合物,而高密度脂蛋白(HDL)则不能。人主动脉硫酸软骨素—PG(CS—PG)、硫酸皮肤素—硫酸软骨素—PG(DS—CS—PG)与血清形成不溶性复合物的曲线类型不同,后者的类型似有利于DS—CS—PG与血清脂蛋白结合从而使之在动脉壁沉积。     

Isolation of serum chylomicrons prior to density gradient ultracentrifugation of other serum lipoprotein classes

A method for the removal of serum chylomicrons before density gradient ultracentrifugation of the other serum lipoproteins using an SW 41 swinging bucket rotor is presented. In a preliminary spin, the chylomicrons with an Sf greater than 400 X 10(-13) s float to the top of the gradient, whereas the other lipoproteins are retained in the infranatant fraction. After removal of the chylomicrons, the other serum lipoproteins are subsequently fractionated by isopycnic density gradient ultracentrifugation. Analysis of the separated lipoprotein fractions suggested that this procedure permits isolation of a chylomicron fraction consisting solely of chylomicrons but that the very low density lipoprotein fraction subsequently isolated also contains chylomicrons or chylomicron remnants with an Sf less than 400 X 10(-13) s, and that there is considerable overlap in flotation rate and particle size of very low density lipoproteins and chylomicrons.     

Radioimmunoassay of arginine-rich apolipoprotein of rat serum.

A double-antibody radioimmunoassay was developed for quantification of rat arginine-rich apolipoprotein in sodium decyl sulfate. Arginine-rich protein, labeled with 125I by the chloramine-T method, had the same chromatographic characteristics on Sephadex G-200 as unlabeled arginine-rich protein and up to 70% of 125I-labeled arginine-rich protein was precipitated by antisera to arginine-rich protein in rabbits. The assay is sensitive at the level of 1-10 ng and has intraassay and interassay coefficients of variation of 5.4 and 6.8%, respectively. The specificity of the assay was established by competitive displacement of 125I-labeled arginine-rich protein from its antiserum by arginine-rich protein and lipoproteins containing this protein, but not by rat albumin or other purified apolipoproteins. Immunoreactivity of rat serum and lipoproteins was complete as demonstrated by comparison with their delipidated form. The accuracy of the immunoassay was further substantiated by comparison with the amount of arginine-rich protein in chromatographic fractions of total apoprotein of very low and high density lipoproteins, and by recovery experiments in ultracentrifugally separated fractions of serum. In contrast to an immunoassay reported previously for rat apo A-I, sodium decyl sulfate was not required for complete immunoreactivity of serum and lipoproteins. The inclusion of sodium decyl sulfate (9 mM final concentration) was necessary, however, for stability of labeled and unlabeled preparations of arginine-rich protein. Content (weight %, means values +/- S.D.), of immunoassayable arginine-rich protein in isolated lipoproteins was 15 +/- 1.5% in very density lipoproteins; 6.8% in low density lipoproteins (1.02 less than d less than 1.04 g/m); 7.1 +/- 0.3% in high density lipoproteins; and 4.8 +/- 0.5% in lymph chylomicrons. Concentration in whole serum was 18.1 +/- 1.4 and 20.4 +/- 2.3 mg/dl for male and female rats, respectively. Only about 55% of arginine-rich protein was recovered in the major lipoprotein classes and about 40% was in "lipoprotein-free" serum (d greater than 1.25 g/ml). Among the lipoproteins, the high density lipoprotein fraction contained twice the amount of arginine-rich protein recovered in very low or low density lipoproteins (26.6 vs. 13.5 and 13.4%, respectively). The significance of the large amount of arginine-rich protein in the 1.25 g/ml infranatant fraction is not apparent. Although repetitive centrifugation did not alter the amount recovered in this fraction, the possibility of an artifact induced by centrifugation and high salt concentration cannot be excluded.     

A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.     

Lipid composition and cholesterol esterification in serum lipoprotein fraction of the horse, Equus caballus.

1. Changes in lipid components of lipoproteins during incubation of horse serum at 37 degrees C were investigated. In non-incubated serum, cholesterol and lecithin existed predominantly in alpha-lipoprotein or in high-density lipoprotein (HDL). Lysolecithin was mainly associated with the fraction with density above 1.21. 2. When serum was separated into alpha- and beta-lipoproteins by the heparin precipitation method after 1 hr incubation, the decrease in alpha-lipoprotein free cholesterol and lecithin was about four times that in beta-lipoprotein counterparts. 3. When serum lipoproteins were separated by ultracentrifugation, the decrease in each lipoprotein free cholesterol was closely paralleled with that in lecithin. 4. HDL appeared to be a preferential substrate for the lecithin: cholesterol acyltransferase reaction. 5. Disc electrophoretic patterns indicated significant differences in the composition of horse serum lipoproteins from those of human and rat.     

Effect of trypsin treatment on the heparin- and receptor-binding properties of human plasma low-density lipoproteins

The effect of trypsin treatment on the heparin- and receptor-binding properties of human plasma low-density lipoproteins (LDL) was examined. LDL were treated with trypsin (2% by weight) for 16 h at 37 degrees C, and the trypsinized core particles (T-LDL) were isolated by gel permeation chromatography on Sepharose CL-4B. Trypsin degraded the apolipoprotein B moiety (Mr = 550,000) of LDL into numerous peptides of Mr less than 110,000, resulting in the release of 25% +/- 5% (n = 6) of its surface-associated protein. Relative to LDL, T-LDL had an increased phospholipid/protein ratio, decreased flotation density and alpha-helical structure, and increased fluidity of the surface and core constituents. Compared to LDL, T-LDL showed a 60% decreased capacity to suppress [1-14C]acetate incorporation into cellular sterols consistent with decreased binding to the LDL receptor. In contrast, T-LDL showed an enhanced capacity to form soluble complexes with heparin in the absence and presence of 2 mM Ca2+. Between 5 and 25 mM Ca2+, both LDL and T-LDL were maximally precipitated by heparin; the stoichiometry of the insoluble complexes (uronic acid/phospholipid, w/w) was 0.054 +/- 0.004 and 0.055 +/- 0.005 (n = 18) for LDL and T-LDL, respectively. Thus, trypsin treatment significantly diminished the lipoprotein's interaction with cells but not with heparin. This finding suggests that proteolysis may decrease receptor-mediated uptake of LDL without diminishing the lipoprotein's reactivity with acellular components of the arterial wall.     

Removal of apoprotein-B-containing lipoproteins by plasmapheresis using immobilized phosphorylcholine-binding protein affinity adsorbent

Rat serum phosphorylcholine binding protein was earlier shown to bind lipoproteins containing apoproteins B and E from human very low and low density lipoproteins. The present studies were undertaken to show the effectiveness of rat serum phosphorylcholine-binding protein immobilized on Sepharose affinity column to remove apoprotein-B-containing lipoproteins from normal and hypercholesterolemic rabbit plasma, when used in a plasmapheresis system. The maximum in vitro binding of very low and low density lipoproteins from hypercholesterolemic rabbit plasma to the affinity adsorbent was Ca2+ dependent, and the cholesterol bound to the column at the optimum calcium concentration (2.5 mM) was 21% of the total plasma cholesterol applied. The in vivo binding of total cholesterol from normal and hypercholesterolemic rabbit plasma during plasmapheresis ranged from 0.22 to 7.7%. Total mass of cholesterol bound ranged from 3.86 and 27.52 mg at plasma cholesterol concentrations 13.8 and 282 mg/dL, respectively. Most (greater than 95%) of the bound cholesterol was associated with very low and low density lipoproteins. These studies show the ability of immobilized rat serum phosphorylcholine-binding protein to lower the atherogenic apoprotein-B-containing lipoproteins from plasma of hypercholesterolemic rabbits.     

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.     

Effects of protamine on blood lipoproteins in the early period of hypercholesterolemia in rabbits

The content of protein, cholesterol and triglycerides in chylomicrons and different classes of blood serum lipoproteins was studied under protamine action in early (1 month) hypercholesterolemia. Except the low density lipoproteins the amount of triglycerides in the particles studied was practically unchanged. The content of proteins in all classes of lipoproteins was greatly increased and this may indicate that protamine activates the blood lipoprotein system. The enrichment of lipoproteins with cholesterol may be taken as being a result of protamine action in early hypercholesterolemia. The peculiar feature in the effect of protamine on mentioned lipoprotein components was that the protein/cholesterol ratio didn't change in chylomicrons, decreased in very low density lipoproteins and low density lipoproteins and increased in high density lipoproteins as compared with controls.     

Plasma lipoprotein and platelet function after heparin injection: studies in normal fasted and postprandial and in type V hyperlipoproteinemic subjects

The effect of heparin injection (50 IU/kg body weight) on plasma lipoprotein concentration and composition as well as on platelet aggregation and 14C-serotonin release was studied in normal fasted subjects, normal subjects 4 hr after a fatty meal (postprandial state), and in primary type V hyperlipoproteinemic patients. Heparin injection resulted in a reduction in plasma triglyceride, cholesterol, and phospholipids as well as in the inhibition of platelet function in either the presence or the absence of the plasma environment. Heparin injection resulted in catabolism of triglyceride-rich lipoproteins and increment of cholesterol and protein in the high-density lipoprotein (HDL) density range. In fasted normal subjects, very-low-density lipoprotein (VLDL) was reduced by 50%; in the postprandial state, both VLDL and chylomicrons decreased similarly; but in phenotype V hyperlipoproteinemia, only chylomicrons (but not VLDL) degraded. Heparin injection also caused increased electrophoretic mobility of plasma lipoprotein. Upon incubation of similar lipoprotein concentration, derived before and after heparin injection, with normal washed platelets, we found that in all the groups all the lipoproteins (except HDL) derived after heparin injection caused reduction in platelet activity. High-density lipoproteins derived after heparin injection, especially from type V hyperlipoproteinemic subjects, increased normal platelet activity, and this probably represents an effect of chylomicron remnant particles in the HDL density range. Our study thus demonstrates altered composition and concentration of plasma lipoprotein after heparin injection and may suggest the appearance of remnant particles with atherogenic properties.     

人动脉壁蛋白聚糖与血清脂蛋白形成的不溶性复合物

本文报道了人主动脉壁中正常及异常(脂纹,FS)区的三种蛋白聚糖(PG)即:硫酸软骨素PG(CSPG)、硫酸皮肤素—硫酸软骨素PG(DSCSPG)及硫酸乙酰肝素PG(HSPG)与血清极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)所形成的不溶性复合物。在30mmol/L Ca~(2+)对,三种PG都能与这两种脂蛋白形成不溶性复合物,随放置时间的增加,形成的复合物都发生解离,但其复合物形成的曲线及解离程度明显不同。DSCSPG与CSPG比较,前者与两种脂蛋白更易形成不溶性复合物且不易解离。HSPG与两种脂蛋白形成不溶性复合物所需时间远大于CSPG及DSCSPG。FS区及正常区三种PG形成复合物曲线类型相似,异常区CSPG、DSCSPG与VLDL形成的复合物量低于正常区的相应PG,而与VLDL则高于正常区的相应PG。异常区的HSPG与两种脂蛋白形成不溶性复合物的量均高于正常区。     

Amino acid composition of the proteins from chylomicrons and human serum lipoproteins

By a combination of polyanion precipitation and ultracentrifugation, chylomicrons, very low density, low density, and high density lipoproteins have been isolated from human serum as discrete classes free from contamination with any other major class of lipoprotein or protein. After removal of the lipid, the proteins from each class were hydrolyzed and their amino acid compositions were determined by use of the amino acid analyzer. Application of the "t" test to the concentrations of amino acid residues showed that the amino acid composition of the proteins from each of these lipoprotein classes differs significantly from class to class. However, when the logarithms of the moles of amino acid residues are plotted, there are similarities in the amino acid "profiles" between the chylomicrons and high density lipoproteins on the one hand, and between the very low density and low density lipoproteins on the other. The differences in amino acid composition between the lipoproteins suggest that any metabolic interconversions between them probably do not occur by simple lipolysis.     

Chloroquine-induced interference with degradation of serum lipoproteins in rat liver, studied in vivo and in vitro.

The effect of chloroquine, an inhibitor of certain lysosomal enzymes including cathepsin B (EC 3.4.22.1), on the degradation of serum lipoproteins in rat liver was studied in vivo and in liver homogenates. Chloroquine had no effect on the clearance from the circulation of 125I-labeled rat or human very low density lipoproteins or human low density lipoproteins. Pretreatment with chloroquine for 3 h, resulted in a 2-2.5 fold increase in 125i-labeled very low density lipoprotein recovered in the liver 45 min after injection of the homologous and heterologous lipoproteins. This effect was evident on both the 125I-labeled protein and 125I-labeled lipid moiety. 30 min after the injection of [3H]-cholesterol linoleate-labeled very low density lipoproteins, 70% of the injected label was recovered in the liver, both in control and chloroquine-treated rats. Since the perl and 20% in the experimental group, it was concluded that chloroquine interferes with the hydrolysis of [3H]cholesterol linoleate. Following injection of 125I-labeled human low density lipoproteins only 4% of the injected lipoprotein was recovered in the liver of control rats and not more than 10% after chloroquine treatment, when about 50% had been cleared from the circulation. Hence, while very low density lipoprotein protein and cholesterol ester are catabolized in the liver, the catabolism of low density lipoproteins occurs mainly in extra-hepatic tissues. Using post-nuclear liver suprnatant, optimal degradation of various serum lipoproteins was found at pH 4.4, and chloroquine inhibited their degradation. Degradation of very low density and low density lipoproteins was completely inhibited at 0.05 M chloroquine, while less pronounced inhibition was seen with high density lipoproteins, apolipoproteins and apolipoprotein AI. These results indicate that liver acid hydrolases in vivo participate in the degradation of serum lipoproteins. Cathepsin B is apparently responsible for the degradation of aplipoprotein B, while other cathepsins might also be active in the degradation of this and the other apolipoproteins.     

The kinetics of binding of serum lipoproteins by immobilized heparin.

A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.     

Inhibition of the binding of low density lipoproteins to liver membrane receptors by rat serum phosphorylcholine binding protein

Rat serum phosphorylcholine binding protein (PCBP) is characterized by its Ca2+ dependent property to bind phosphorylcholine ligand. PCBP immobilized on sepharose has been shown to selectively bind human plasma apo B and E containing lipoproteins. The present report describes an inhibitory effect of PCBP on the binding of human 125I-LDL to LDL receptors on estradiol treated rat liver membranes. Pre-incubation of liver membranes with PCBP did not affect the binding of 125I-LDL to the membranes. Gel filtration analysis of the incubation products from the LDL-receptor assay showed a concentration dependent binding of 125I-PCBP to LDL. The inhibitory effect of PCBP is likely due to the formation of LDL-PCBP complex and not due to the binding of PCBP to the LDL receptor site.     

Mersalyl, a sulfhydryl reagent, alters the solubility of myosin and cytoskeletal proteins of human platelets

We have examined the effect of a mercurial sulfhydryl reagent, mersalyl, on the protein composition of cytoskeletons by SDS-polyacrylamide gel electrophoresis after treatment of human platelets with Triton X-100 (Triton) containing mersalyl and Ca2+, and have found that mersalyl alters the protein composition of cytoskeletons in a Ca2+-dependent manner. At 1 X 10(-7) M Ca2+, 0.2 mM mersalyl, which represents approximately the equivalent amount of sulfhydryl of platelet suspensions that we used, specifically made myosin insoluble. The amount of myosin in Triton-mersalyl residues was increased by increasing the Ca2+ concentration of Triton lysis buffer. Actin-binding protein, 235 kDa polypeptide and alpha-actinin-like protein were decreased in Triton residues by mersalyl at Ca2+ concentrations less than 1 X 10(-7) M, while these polypeptides in Triton residues were increased by mersalyl in the presence of more than 2 X 10(-7) M Ca2+. Electron microscopic study revealed the presence of thick filaments with an appearance similar to that of the thick filaments of platelet myosin. Thus, the modification with mersalyl of sulfhydryls of platelet polypeptides along with changes in Ca2+ concentrations within a physiological range leads to changes in solubility of, and filament formation of, myosin, actin and other cytoskeletal proteins.     

Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 microM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 microM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2(+)-hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2(+)-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.