Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein.

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 microM 5'-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 +/- 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 +/- 0.7 nM) to one site of intermediate affinity (KD = 0.52 +/- 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor

The binding of iodinated basic fibroblast growth factor (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.     

High affinity binding sites for basic fibroblast growth factor in rat hepatic plasma membranes

The binding of [125I]-recombinant basic FGF (rec bFGF) to rat hepatic plasma membranes was investigated. [125I] rec bFGF bound to an apparent single class of high affinity binding sites (KD = 69 pM; Bmax = 9.61 fmoles/mg proteins). The absence of low affinity sites was confirmed by the inability of sulphated polysaccharides and heparinase to interfere with FGF binding. A good correlation existed between the ability of bovine pituitary-derived bFGF, rec bFGF and bovine brain-derived aFGF to displace [125I]rec bFGF from these binding sites and their in vitro potency on bovine aortic endothelial cell proliferation.     

Characterization of cardiac Na+/Ca(2+)-exchange by site-directed polyclonal antibodies.

Cardiac Na+/Ca(2+)-exchange is an integral membrane protein consisting of approx. 970 amino acids with as many as 12 membrane-spanning and 11 extramembranal regions (Nicoll, D.A., Lognoni, S. and Philipson, K.D. (1985) Science 250, 562-565). Based upon primary sequence information, 3 amino-acid sequences located in either extramembranal segment a or f, consisting largely of acidic amino-acids, were selected for the production of synthetic peptides. The peptides were cross-linked to carrier ovalbumin and used to generate site-directed polyclonal antibodies (sd-Ab). Western blot analysis of bovine cardiac sarcolemmal (SL) proteins demonstrated that sd-Ab against segment a and 1 against loop f recognized a 70 kDa protein and a lower molecular mass band at 55 kDa under reducing conditions. A different loop f sd-Ab failed to recognize the 70 kDa protein but did associate with a 120, 65 and 55 kDa protein under the same conditions. Under non-reducing conditions, antibodies to all three peptides recognized the 65 kDa protein. All sd-Ab were blocked by addition of their respective peptides and were not inhibited by either of the other peptides. A sd-Ab against loop f was immobilized to an affinity support matrix and used to immunoprecipitate detergent solubilized cardiac SL vesicle protein. Immunoprecipitated protein was reconstituted into proteoliposomes which demonstrated Na+/Ca(2+)-exchange activity. Immunoprecipitated protein cross-reacted with sd-Ab against all three peptides with bands at 120, 70 and 55 kDa on Western blots. Tryptic digests of native SL vesicles abolished recognition of segment a sd-Ab for SL proteins while having little or no affect on reactivity to the protein by both sd-Ab against loop f. Digestion of the SL vesicle protein with endoproteinase Arg C did not alter sd-Ab recognition. The results suggest that specific domains of the cardiac Na+/Ca(2+)-exchanger depending upon the conformation of the protein, may not be available for antibody binding. The 70 kDa polypeptide appears to include the N-terminal region of the protein and what is believed to be a large cytoplasmic extramembranal loop. Limited proteolysis by trypsin and endoproteinase Arg C yielded results consistent with the model which places the N-terminus of the protein on the extracellular surface and a large extramembranal segment (loop f) on the cytoplasmic side of the SL membrane.     

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.     

Characterization of calpain I-binding proteins in human erythrocyte plasma membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).     

Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease

Neurofibrillary tangles (NFT) are abnormal filamentous inclusions that develop in neurons in Alzheimer disease and other disorders. When neurons die, the neurofibrillary tangles that persist in the extracellular space show ultrastructural and antigenic changes. Both intra- and extracellular NFT have recently been shown to contain heparan sulfate proteoglycans (HSPGs). HSPGs are also present in other amyloid deposits in the brain and in systemic amyloidoses. Basic fibroblast growth factor (bFGF) is a heparin binding growth factor which is involved in angiogenesis and also has neurite promoting activity. We now report that bFGF binds avidly to extracellular NFT. Alz-50, a monoclonal antibody (MAb) to an abnormal form of tau and bFGF binding label mutually exclusive subpopulations of neurofibrillary tangles. bFGF binding is abolished by heparinase or heparitinase treatment and therefore is most likely based on the presence of HSPG. Binding of bFGF is a specific and sensitive morphological method to distinguish intra- from extracellular NFT. As intracellular NFT, which also contain HSPGs, are not labeled by bFGF binding, this finding also suggests that HSPGs are modified when the NFT become extracellular.     

Interaction of alpha-crystallin with lens plasma membranes. Affinity for MP26

The binding of the major water-soluble lens protein alpha-crystallin to the lens plasma membrane has been investigated by reassociating purified alpha-crystallin with alpha-crystallin-depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. alpha-Crystallin reassociates at high affinity (Kd = 13 X 10(-8)M) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of alpha-crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for alpha-crystallin binding. Labeling experiments using (trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that alpha-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized alpha-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of alpha-crystallin aggregates does not take place in a membrane-bound mode.     

Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.     

Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3

We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.     

Synapsin I-mediated interaction of brain spectrin with synaptic vesicles

We have established a new binding assay in which 125I-labeled synaptic vesicles are incubated with brain spectrin covalently immobilized on cellulosic membranes in a microfiltration apparatus. We obtained saturable, high affinity, salt- (optimum at 50-70 mM NaCl) and pH- (optimum at pH 7.5-7.8) dependent binding. Nonlinear regression analysis of the binding isotherm indicated one site binding with a Kd = 59 micrograms/ml and a maximal binding capacity = 1.9 micrograms vesicle protein per microgram spectrin. The fact that the binding of spectrin was via synapsin was demonstrated in three ways. (a) Binding of synaptic vesicles to immobilized spectrin was eliminated by prior extraction with 1 M KCl. When the peripheral membrane proteins in the 1 M KCl extract were separated by SDS-PAGE, transferred to nitrocellulose paper and incubated with 125I-brain spectrin, 96% of the total radioactivity was associated with five polypeptides of 80, 75, 69, 64, and 40 kD. All five polypeptides reacted with an anti-synapsin I polyclonal antibody, and the 80- and 75-kD polypeptides comigrated with authentic synapsin Ia and synapsin Ib. The 69- and 64-kD polypeptides are either proteolytic fragments of synapsin I or represent synapsin IIa and synapsin IIb. (b) Pure synapsin I was capable of competitively inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin with a Kl = 46 nM. (c) Fab fragments of anti-synapsin I were capable of inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin. These three observations clearly establish that synapsin I is a primary receptor for brain spectrin on the cytoplasmic surface of the synaptic vesicle membrane.     

Roles of proteins in cation/membrane interactions of isolated rat cardiac sarcolemmal vesicles

To ascertain the roles of the membrane proteins in cation/sarcolemmal membrane binding, isolated rat cardiac sarcolemmal vesicles were extensively treated with Protease (S. aureus strain V.8). SDS-gel electrophoresis, protein and phosphate analysis confirmed that at least 20–22% of the protein, but none of the phospholipid, was solubilized by this procedure, and that the remaining membrane proteins were extensively hydrolyzed into small fragments. The cation binding properties of the treated vesicles were then examined by analyzing their aggregation behavior. The results demonstrate that this procedure had no effect on the selectivity series for di- and trivalent cation binding, or the divalent cation-induced aggregation behavior of the sarcolemmal vesicles at different pHs, indicating that proteins are probably not involved in these interactions and cannot be the low affinity cation binding sites previously observed [21, 22]. It did, however, change the pH at which protons induced sarcolemmal vesicle aggregation, suggesting a possible role for proteins in these processes. Protease treatment also modified the effects of fluorescamine labelling on divalent cation-induced vesicle aggregation, indicating that the NH, groups being labelled with fluorescamine are located on the sarcolemmal proteins. Together, these results support the hypothesis that di- and trivalent cation binding to the sarcolemmal membrane is largely determined by lipid/lipid and/or lipid/carbohydrate interactions within the plane of the sarcolemmal membrane, and that membrane proteins may exert an influence on these interactions, but only under very specialized conditions.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - HEPES N-2-Hydroxyethylpiperizine-N-2- ethanesulfonic acid - CHES 2(N-Cyclohexylamino) ethanesulfonic acid - DTT DL-Dithiothreitol - PMSF Phenylmethyl-sulfonyl fluoride     

Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro

We have established an in vitro assay to characterize the binding of endocytic carrier vesicles to microtubules. Magnetic beads coated with microtubules were used as an affinity matrix. A fraction from nocodazole-treated cells enriched in endocytic carrier vesicles, labeled with internalized horseradish peroxidase, was used in the binding experiments. Binding of the endocytic carrier vesicles to microtubules in vitro was cytosol-dependent. This activity of cytosolic factors was saturable, heat-sensitive, and insensitive to N-ethyl-maleimide. Binding was sensitive to GTP and ATP. Addition of neuronal microtubule-associated proteins completely abolished binding of the endocytic organelles to microtubules. This binding was independent of the cytosolic microtubule-based motor proteins kinesin and cytoplasmic dynein, since cytosol depleted of these proteins remained fully active. Microtubule-binding proteins from HeLa cells, however, stimulated the interaction of endocytic carrier vesicles with microtubules. Trypsinized vesicles could no longer bind to microtubules in the presence of cytosol. These results suggest that cytosolic microtubule-binding proteins, other than the known microtubule-based motor proteins, as well as membrane proteins are involved in the nucleotide-dependent interaction of endocytic carrier vesicles with microtubules.     

Transforming growth factor beta 1 stimulates the production of basic fibroblast growth factor binding proteoglycans in Balb/c3T3 cells.

Basic fibroblast growth factor (bFGF) binds to cell surface receptors and to heparin sulfate proteoglycans. Heparan sulfate binding may limit bFGF degradation and be an obligatory step for bFGF cell interaction. Transforming growth factor-beta 1 (TGF-beta 1) is a potent regulator of proteoglycan production and composition. The possibility that TGF-beta 1 synergistically regulates bFGF activity by altering bFGF-proteoglycan interactions was investigated. TGF-beta 1 increased 125I-bFGF binding to the extracellular matrix (ECM) of Balb/c3T3 cells 2-4-fold by increasing the number of bFGF binding sites. Increased bFGF binding correlated with a 2-5-fold increase in the production of sulfated proteoglycans, including heparan sulfate proteoglycans. TGF-beta 1 selectively stimulated production of high molecular mass proteoglycans (190-300 kDa) in conditioned medium and stimulated all proteoglycans in ECM. 125I-bFGF bound to TGF-beta 1 induced proteoglycans immobilized onto cationic nylon filters. Furthermore, ECM isolated from TGF-beta 1-treated cells incorporated more mitogenically active bFGF than native ECM. The mitogenic potential of the ECM was significantly reduced by treatment with heparinase. These results suggest that the ability of TGF-beta 1 to stimulate binding of bFGF to ECM, increase ECM heparan sulfate proteoglycan, and potentiate the mitogenic activity of bFGF are linked. Thus one aspect of TGF-beta 1/bFGF synergy may involve modulation of the ECM.     

High affinity binding of an N-terminal myristoylated p60src peptide

N-Myristoyl and non-myristoyl peptides corresponding to the N terminus of p60src were used to examine whether N-myristoylation facilitates the binding of p60src to specific protein sites at the plasma membrane. We discovered high affinity protein acceptor sites (Kd = 2.7 nM) to a 15-amino acid N-myristoylated N-terminal p60src peptide in red cell membrane vesicles. Binding was not competed by the non-myristoylated analog of the peptide nor by shorter N-myristoyl src peptides and peptides homologous to the N terminus of other N-myristoylated proteins. Binding was not evident after treatment of vesicles with proteolytic enzymes. Raising the salt concentration of the buffer to 50 mM NaCl caused an apparent inhibition of binding. However, no significant effect of salt was observed on the off-rate of bound ligand under these conditions. The results indicate the existence of N-myristoyl-dependent p60src protein acceptor sites at or near the plasma membrane/skeleton interface of red cells which could be responsible for the localization of p60src to this region and may represent new regulatory components for p60src-mediated tyrosine kinase activity.     

Regulation of basic fibroblast growth factor binding and activity by cell density and heparan sulfate

The role of cell density in modulating basic fibroblast growth factor binding and activity was investigated. A primary corneal stromal fibroblast cell culture system was used, since these cells do not constitutively express heparan sulfate proteoglycans in vivo except after injury. A 3-5-fold reduction in bFGF binding per cell was observed as cell density increased from 1000 to 35,000 cells/cm2. The cell density-dependent change in bFGF binding was not the result of altered FGFR expression as determined by equilibrium binding experiments and by immunoblot analysis. However, bFGF-cell surface receptor binding affinities were measured to be 10-20-fold higher at low cell densities than at intermediate and high cell density. bFGF-induced cell proliferation was also cell density-dependent, with maximal stimulation of proliferation 190-280% greater at intermediate densities (15,000 cells/cm2) than at other cell densities. This effect was specific to bFGF as serum, epidermal growth factor, and transforming growth factor-beta did not exhibit the same density-dependent profile. Further, heparan sulfate proteoglycans and, specifically, syndecan-4 were implicated as the modulator of bFGF binding and activity. Pretreatment of cell cultures with heparinase resulted in reduced bFGF binding to the cells and abrogated bFGF induced proliferation. These data suggest a mechanism by which cell density regulates heparan sulfate proteoglycan expression and modulates the cellular response to bFGF. Modulation of heparan sulfate proteoglycan expression might be an important aspect of the regulation of stromal cell migration and proliferation during wound healing.     

Binding of a mitochondrial presequence to natural and artificial membranes: role of surface potential.

The binding of a synthetic mitochondrial presequence to large, negatively charged, unilamellar vesicles and to unenergized yeast mitochondria has been measured. The presequence, which corresponds to the amino-terminal 25 residues of the yeast cytochrome oxidase subunit IV precursor, was labeled with a fluorescent probe and used to examine the importance of the surface potentials of membranes on the interactions with the presequence. Binding of the fluorescent presequence to the membranes was determined by measuring a decrease in the fluorescence emission of the bound presequence. Binding both to the vesicles and to the mitochondria could be described as a simple partitioning of the presequence between the aqueous and lipid phases. The partitioning was found to depend on the ionic strength of the medium, and the Gouy-Chapman theory could be used to describe the partitioning at various ionic strengths. Application of the theory allowed the determination of an apparent charge on the presequence (+2.31 +/- 0.25), salt-independent apparent partition coefficients for vesicles (99 +/- 84 M-1) and for unenergized mitochondria (14.5 +/- 3.6 L g-1), and an estimated charge density for the mitochondrial outer membrane (-0.0124 +/- 0.0016 C m-2). This study shows that electrostatic effects are significant for the binding of a mitochondrial presequence both to lipid vesicles and to mitochondria, the natural target membrane of the presequence. The accumulation of positively charged presequences at the negative mitochondrial surface and the subsequent partitioning of the presequences directly into the mitochondrial outer membrane probably represent early steps in the translocation of precursor proteins into mitochondria.     

ATP-dependent calcium transport in cardiac sarcolemmal membrane vesicles

Cardiac sarcolemmal (SL) membrane vesicles accumulated Ca in the presence of ATP. The accumulated Ca was released by osmotic shock and by the Ca ionophore A23187, indicating that the Ca had been transported into the vesicle interior. Ca uptake by the SL vesicles was not inhibited by ruthenium red, 2,4-dinitrophenol, carbonyl cyanide $\text{m}$-chlorophenyl hydrazone, of NaN₃, agents that are known to inhibit mitochondrial Ca transport activity. In contrast to the behavior of cardiac sarcoplasmic reticulum, Ca accumulation by the SL vesicles was not stimulated by oxalate and could not driven by p-nitrophenylphosphate hydrolysis. NaCl inhibited ATP-dependent Ca uptake by the SL vesicles. This effect was shown to be due to a stimulation of Ca efflux by Na, mediated by the sarcolemmal NaCa exchange system. The results provide conclusive evidence for the presence of an ATP-dependent Ca “pump” in the cardiac SL membrane.     

Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.     

Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells

Bovine capillary endothelial (BCE) cells were incubated at 4 degrees C with 5 ng/ml 125I-basic fibroblast growth factor (bFGF) to equilibrate 125I-bFGF with high affinity cell surface receptors and low affinity matrix binding sites. 67% of the added 125I-bFGF bound to the matrix and 7% bound to receptors. The fate of bound bFGF was followed after cells were incubated in bFGF-free medium and were shifted to 37 degrees C to restore cell metabolism. 125I-bFGF bound to receptors decreased rapidly while the amount of 125I-bFGF bound to matrix was reduced more slowly. The rapid decrease in receptor-bound 125I-bFGF appeared to be due to a down-regulation of bFGF receptors; cells that had been treated for 5 h with bFGF had 60% fewer high affinity receptors than untreated cells. Despite the initial high level of 125I-bFGF binding to matrix, most of this 125I-bFGF was mobilized and metabolized by the cells. 125I-bFGF was internalized by the cells at 37 degrees C, leading to a constant accumulation of 125I-bFGF within the cell. Internalized bFGF was rapidly cleaved from an 18-kD form to a 16-kD form. The 16-kD form was more slowly degraded with a half-life of approximately 8 h. Degradation of internalized 125I-bFGF was inhibited by chloroquine, suggesting that the digestion occurred in a lysosomal compartment. The role of matrix binding sites in the internalization process was investigated. Binding to matrix sites seemed not to be directly involved in the internalization process, since addition of heparin at a concentration that blocked 95% of the binding to matrix had no effect on the initial rate of internalization of bFGF. BCE cells also released a substance that competed for the binding of bFGF to matrix but not to receptors. This substance bound to DEAE-cellulose and was sensitive to heparinase treatment, suggesting that it was a heparinlike molecule. Thus, heparinlike molecules produced by BCE cells can modulate the cellular interaction with bFGF. Matrix-associated heparinlike molecules bind bFGF which can later be metabolized by the cell, and secreted heparinlike molecules release bFGF from matrices.