Regulation of eye derived growth factor binding to membranes by light, ATP or GTP in photoreceptor outer segments.

Eye derived growth factor II (EDGF II), the retinal form of acidic fibroblast growth factor (aFGF) is present in rod outer segments (ROS) purified in the dark, which display higher EDGF specific activity than all other parts of the retina. EDGF binds to ROS disc membranes upon illumination. This binding is not reversible in the dark. ATP, but not GTP, readily releases EDGF from either dark-adapted or previously bleached ROS. The release of EDGF activity from ROS membranes would require a phosphorylation mechanism since AMP-PNP, an ATP analogue, is not efficient. ROS membranes compete with cellular EDGF receptors of retinal pigment epithelial cells in vitro for the binding of labelled EDGF II, suggesting that they also possess specific binding sites. These data suggest that EDGF II is involved in photoreceptor cell biology.     

Insulin-like growth factor I receptors in retinal rod outer segments

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.     

Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissues

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Presence of basic fibroblast growth factor receptors in bovine brain membranes

We described a protocol for purification of bovine brain membranes suitable to study the binding of iodinated basic fibroblast growth factor (FGF) to bovine brain membrane preparation. The binding of 125I basic FGF to brain membranes reached equilibrium within 30 min at 20 degrees C, was reversible, and displaced by an excess of unlabeled basic FGF. Scatchard analysis of the data revealed that two classes of binding sites could be detected with an apparent Kd of 30 pM and a capacity of 0.24 pmol/mg of membrane proteins for the high affinity binding site and Kd of 3 nM with a capacity of 51 pmol/mg of membrane proteins for the low affinity binding site. Cross-linking experiments of labeled basic FGF to brain membrane receptor yield the formation of a single major complex with an apparent molecular mass of 170 kDa which is similar to the value obtained for the high affinity binding site for basic FGF on target cells in tissue culture. Hence these data present the first biochemical evidence suggesting that membrane purified from bovine brain contain two classes of specific binding sites for basic FGF and confirm results described with cells grown in vitro.     

Solubilization of the omega-conotoxin receptor associated with voltage-sensitive calcium channels from bovine brain

The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes.     

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 1. Probing of the nucleotide sites by Naphthoyl-ATP, a fluorescent nontransportable analogue of ATP

The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.     

Cell type and tissue distribution of the fibroblast growth factor receptor

A receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165-kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I-aFGF to its cell surface receptor was specifically blocked by a 100-fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I-aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I-aFGF binding to cell- and tissue-derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I-aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.     

Effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) on the binding of 3H-quinuclidinyl benzilate to striatal cholinergic muscarinic receptors

The effects of Pro-Leu-Gly-NH2 (melanotropin release inhibiting factor, MIF) and its analog, cyclo (Leu-Gly) on the mouse and rat striatal cholinergic muscarinic receptors labeled with 3H-quinuclidinyl benzilate (QNB) were investigated. 3H-QNB bound to the rat striatal muscarinic receptors at a single high affinity site with receptor density (Bmax value) of 1200 fmol per mg protein and an apparent dissociation constant (Kd value) of 53.5 pM. At 140 pM concentration of 3H-QNB, the specific binding to the receptors was 724 fmol per mg protein. MIF in a concentration range of 10(-9) to 10(-4) M did not alter the binding of 3H-QNB but at 10(-3) M decreased the binding by 25%. Cyclo (Leu-Gly), on the other hand, in the concentration range of 10(-9) to 10(-3) M had no effect on the binding of 3H-QNB. A single injection of MIF (3 or 10 mg/kg IP) to rats did not alter the Bmax or the Kd value of 3H-QNB to bind to the striatal membranes. 3H-QNB bound to the mouse striatal muscarinic receptors at a single high affinity site with a Bmax value of 991 fmol/per mg protein and a Kd value of 21 pM. Neither acute administration of MIF (3 or 10 mg/kg IP) nor chronic treatment of the peptide (2, 8 or 32 mg/kg IP, daily for 5 days) to mice could influence the binding of 3H-QNB to the striatal muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

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.     

Phosphorylation of endogenous and exogenous peptides by rat heart sarcolemma

Rat heart plasma membranes contain a calcium-dependent protein kinase which phosphorylates endogenous protein substrates as well as added histones. The major endogenous protein phosphorylated is of 17 kDa on SDS-polyacrylamide gel electrophoresis. Proteins of 85 kDa and 60 kDa were also phosphorylated. Treatment of a rat heart homogenate with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased the recovery of kinase activity in the sarcolemmal membranes by up to 10-fold. The activity in such membranes was no longer calcium dependent. Although several histones were effective substrates for the enzyme, myosin light chain and phosvitin were not phosphorylated. These membranes contain a very active ATP hydrolysing activity which necessitated very brief incubation times to avoid loss of substrate. The membranes also contain cyclic AMP dependent protein kinase activity which is not active unless cyclic AMP is added to the incubations. The calcium dependent endogenous kinase, which is not inhibited by the heat stable inhibitor protein of cyclic AMP-dependent kinase, or by trifluoperazine, has several properties in common with protein kinase C. Preincubation of the sarcolemmal membranes with a high concentration of insulin caused inhibition of the phosphorylation of the endogenous 17 kDa and 85 kDa bands. There was no effect on the phosphorylation of the 60 kDa peptide. This effect of insulin was specific for the hormone and required preincubation of the hormone with the membranes for 20 min.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Characterization of synapsin I binding to small synaptic vesicles

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

The catalytic site is located on subunit I of the ATPase from Halobacterium saccharovorum. A direct photoaffinity labeling study.

Nucleotide-binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of [alpha-32P]ATP. A high-affinity site, located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross-linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP-inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non-catalytic nucleotide-binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1-ATPases which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.