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1.
The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and 89 mV, respectively. These shifts are consistent with the different susceptibilities of Uf to aerobic oxidation in the presence of these anions. Comparison of the reduction potential of Uf at pH 7.0 with those reported for other dinuclear non-heme iron enzymes and various (mu-oxo)diiron model complexes suggest that the potential of Uf is too positive to be consistent with a mu-oxo-bridge in Ufo. The pH dependence of the reduction potentials of Uf (60 mV/pH unit) and the fact that the electron transfer rate increases with decreasing pH indicate a concomitant participation of a proton during the oxidation-reduction process. This process was assigned to the protonation of a terminally bound hydroxide ligand at the Fe(II) center upon reduction of Ufo. Structural implications provided by the electrochemical data indicate that molybdate affects the dinuclear core in a manner that differs from that of phosphate and arsenate. This observation is consistent with previous spectroscopic and biochemical studies.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
G A Baumbach C M Ketcham D E Richardson F W Bazer R M Roberts 《The Journal of biological chemistry》1986,261(27):12869-12878
A pink, high molecular weight form of uteroferrin (Uf) has been isolated from uterine secretions and allantoic fluid of pigs. This protein fraction (denoted FIII) which is relatively stable under physiological conditions of pH, ionic strength, and temperature has a molecular weight of about 80,000, a value approximately twice that of purple Uf (Mr approximately 35,000) isolated from a separate fraction (FIV) by gel filtration. The visible absorption spectrum, EPR signal, and acid phosphatase activity of Uf in FIII are almost identical to those of FIV Uf after the latter has been reduced by 2-mercaptoethanol. However, unlike reduced FIV Uf, the pink, high molecular form does not revert to purple, nor does it show loss of EPR signal and phosphatase activity in the presence of oxygen. In addition, it does not become purple at orthophosphate concentrations which inhibit Uf acid phosphatase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has shown that FIII consists of approximately equal amounts of Uf polypeptides (Mr = 35,000 and 37,000) and a group of three polypeptides (Mr = 40,000, 46,000, and 50,000) antigenically unrelated to Uf. The latter share a common epitope not found on Uf and are probably differentially processed forms of the same protein. FIII can be dissociated by pH conditions below 5.0, by exposure to antibodies raised against Uf or the associated polypeptides, and by sodium dodecyl sulfate at 100 degrees C. The polypeptides in FIII are not therefore linked by disulfide bonds. Treatment with dimethyl suberimidate, however, results in a cross-linked complex (Mr approximately 82,000) consisting of Uf and the associated polypeptides. It is concluded that this high Mr form of Uf is a heterodimer of fully activated Uf and a second polypeptide of unknown function. 相似文献
3.
There is continuing controversy as to whether iron can be exchanged from the purple phosphatase, uteroferrin (Uf), to fetal transferrin (Tf) and whether this process might be of physiological relevance during pregnancy in the pig. Here, iron transfer from Uf to apoTf at pH 7.1 was followed by measuring the loss of acid phosphatase activity from native Uf as a function of incubation conditions and time. In the presence of apoTf and 1 mM ascorbate (but not in the presence of either agent alone), 50% of enzyme activity was lost in about 12 h. Loss of activity was accompanied by bleaching of Uf purple color and the appearance of the characteristic visual absorption spectrum of Fe-Tf. Citrate could replace ascorbate in the reaction. Loss of Uf iron did not occur at pH 5.3, at which pH Tf cannot bind Fe. [59Fe]Uf was prepared and shown to be identical in its enzymatic and physical properties with unmodified Uf. Transfer of 59Fe from Uf to apo-Tf was promoted by conditions identical to those which led to loss of purple color and acid phosphatase activity. However, the results suggested that only one of the two iron atoms at the bi-iron center on Uf was readily lost, and that exchange of the second iron occurred more slowly. Loss of iron made Uf more susceptible to denaturation. A third technique, quantitation of the g' = 4.3 signal of iron specifically bound to Tf by EPR, was also tested as a means assaying accumulation of Fe-Tf, but the method was too insensitive to measure the kinetics of iron transfer at physiological protein concentrations. We conclude that iron can be transferred directly from Uf to apoTf in the presence of low molecular weight chelators, and that the process is likely to be of physiological significance. 相似文献
4.
The pH dependence of formation of a peroxidatic intermediate from the reaction of deuteroferriheme with hydrogen peroxide has been determined for the region pH 8.7-10.1 from stopped-flow kinetic studies in which absorbancy changes are observed at heme monomer-dimer isosbestic points. Results are interpreted primarily in terms of the attainment of double "steady-state" concentrations of Michaelis-Menten complex I and peroxidatic intermediate I'. A linear correlation of observed first-order rate constants with alpha, the degree of dissociation of heme dimer, has been demonstrated and nonzero intercepts are obtained. Slopes and intercepts show a linear logarithmic dependence on pH which is interpreted in terms of HO2-participation both in the formation and subsequent (catalatic) decomposition of a peroxidatically active intermediate. General acid catalysis of intermediate formation is indicated from studies in phosphate, arsenate, and citrate buffer at pH 7.4-9.3. It is suggested that such catalysis may be responsible for anomalously high rates of H2O2 decomposition previously observed in phosphate buffer solution. 相似文献
5.
The binuclear iron cluster of uteroferrin in its reduced and enzymatically active pink form is sensitive to a variety or perturbants. Orthophosphate, in the presence or absence of oxygen, rapidly shifts the absorption maximum of pink uteroferrin from 510 to 545 nm, concurrently abolishing the protein's g'av = 1.74 EPR signal. Apparently, therefore, dioxygen is not required for phosphate-induced oxidation of the pink protein's ferrous iron. Pyrophosphate and arsenate produce changes which differ only in degree from those induced by phosphate, suggesting that all of these structurally similar competitive inhibitors bind to a common site. Molybdate, an inhibitor even more potent than phosphate, quantitatively converts the rhombic EPR signal of pink uteroferrin into an axial signal that remains invariant to subsequent additions of phosphate. Thus, there can be inhibition without oxidation, as further evidenced by the complex EPR spectrum of undiminished intensity produced by sulfate. Fluoride, too, induces an axial component in the EPR signal of pink uteroferrin, but at high concentration abolishes the signal entirely. Vanadate also drives the protein to its oxidized, EPR-silent state, serving as an electron acceptor itself to yield the characteristic g' = 2 signal of the vanadyl (VO2+) cation. Remarkably, however, the protein remains pink, demonstrating a dissociation between color and oxidation state. Guanidinium, in contrast, causes a sizeable red shift in the pink protein's absorption maximum without loss of EPR signal intensity, showing dissociation of color and oxidation state in a complementary way. 相似文献
6.
Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding Ki values ranging from 10.5 to 14.1 × 10?6 M in comparison to the corresponding apparent KM (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO4??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO4?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10?4 M), and adenosine triphosphate (1–5 × 10?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca++ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca++ and Mg++ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′2 = 6.14 and for pK′3 = 10.99, at 15°C and phosphate at infinite dilution. 相似文献
7.
The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well. 相似文献
8.
Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation. 总被引:2,自引:0,他引:2
Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin. 相似文献
9.
Phosphate binding to liver alcohol dehydrogenase studied by the rate of alkylation with affinity labels 总被引:1,自引:0,他引:1
In this work we report that phosphate anions interact with the anion binding site of alcohol dehydrogenase from horse liver. In protection experiments against the two affinity labels, iodoacetic acid and bromo-imidazolylpropionic acid, the dissociation constant for the enzyme-phosphate complex at pH 7.0 is, based on total phosphate, found to be 20 +/- 5 mM. The 1,4-piperazinediethanesulfonate anion has a lower affinity for the anion binding site, the dissociation at pH 7.0 being 130 +/- 20 mM. The anion-independent dissociation constants for the reversible enzyme-affinity label complexes are at pH 7.0, 1.35 +/- 0.2 mM for iodoacetic acid and 0.39 +/- 0.05 mM for bromo-imidazolylpropionic acid. These findings have important implications with respect to past and future work on this well known enzyme. 相似文献
10.
Interaction of arsenate with phosphate-transport systems in wild- type and mutant Streptococcus faecalis 总被引:9,自引:7,他引:2
Harold, F. M. (National Jewish Hospital, Denver, Colo.), and J. R. Baarda. Interaction of arsenate with phosphate-transport systems in wild-type and mutant Streptococcus faecalis. J. Bacteriol. 91:2257-2262. 1966.-Arsenate competitively inhibits the growth of Streptococcus faecalis, primarily by competition with phosphate for a common transport system. Arsenate is itself accumulated by the cells; the uptake requires metabolic energy, and the intracellular arsenate level may reach 0.01 m. Cells loaded with arsenate have lost the capacity to take up radioactive glutamate, rubidium, phosphate, or arsenate itself, apparently by the uncoupling of adenosine triphosphate generation. The pH dependence of arsenate uptake is complex. At low concentrations of extracellular arsenate, uptake by the wild-type strain 9790 exhibits a single maximum about pH 8; mutant PT-1, previously shown to be defective in phosphate uptake, takes up essentially no arsenate. At high concentrations of arsenate, uptake by the wild type is bimodal with maxima at pH 5.5 and 9; the uptake curve for mutant PT-1 corresponds to the shoulder in the curve for the wild type. The apparent dissociation constant for arsenate uptake by the wild type is approximately 10(-5)m from pH 5 to 9, whereas that for mutant PT-1 is about 5 x 10(-5) M at pH 5 and rises rapidly with increasing pH. The results confirm the earlier conclusion that the lesion in mutant PT-1 resides in the transport of phosphate and arsenate. It is proposed that the wild type has two distinct transport systems, whereas the mutant has lost the one with alkaline pH optimum. 相似文献
11.
Complex formation between Arsenazo III and Mn2+ and Co2+ at equilibrium has been investigated at pH 7.2, and the stoichiometry and stability of the complexes have been determined. The data indicate that Arsenazo III is suitable for determination of Mn2+ and Co2+ on the micromolar scale. The dissociation constants of the phosphate complexes of Mn2+ and Co2+ at pH 7.2 were estimated with Arsenazo III as 3.6 and 10 mM, respectively. 相似文献
12.
George A. Baumbach Philippa T. K. Saunders Catherine M. Ketcham Fuller W. Bazer R. Michael Roberts 《Molecular and cellular biochemistry》1991,105(2):107-117
Mature uteroferrin (Uf; M = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a M of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man6-4GlcNac2 oligosaccharides found on mature Uf.Abbreviations Uf
Uteroferrin
- ConA
Concanavalin A
- WGA
Wheat Germ Agglutinin
- endoH
endo--N-acetylglucosaminidase H
- SDS
Sodium Dodecyl Sulfate
- SDS-PAGE
polyacrylamide gel electrophoresis in the presence of SDS 相似文献
13.
The nicotianamine molecule is made-to-measure for complexation of metal micronutrients in plants 总被引:8,自引:0,他引:8
The non-proteinogenic amino acid nicotianamine (NA) is ubiquitous among plants. In meristematic tissues it reaches concentrations of about 400mol (g fresh weight)–1. NA forms complexes, among others, with the metal micronutrients (MN) copper, zinc, iron and manganese (logK
MeNA 18.6-8.8). Calculations of the dissociation curves of the metal-NA complexes based on the complex formation constants and on the acid dissociation constants of NA revealed their stability at the neutral or weak alkaline pH of cytoplasm and sieve tube sap. For the Mn-NA complex, dissociation begins at about pH 6.5, for all others dissociation occurs at more acid pHs. Thus, metal-NA complexes could theoretically persist also in the apoplasm and in xylem sap. The octanol water partition coefficient of NA is about 1 and those of its metal complexes are in the range of 0.3–0.4. The reason for this shift is perhaps the negative charge of the complexes. The higher lipophilicity of the free NA indicates that the NA supply to sites of requirement is faster than the removal of the complexes as long as membranes are an integral part of the transport paths. Changing phloem transport rates of MN-NA complexes by manipulation of the cotyledon apoplasm of Ricinus commuais L. suggest a competition of MN for NA at the site(s) of phloem loading. Thus, NA could control MN transport via phloem including recirculation. 相似文献
14.
A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development. 相似文献
15.
The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed. 相似文献
16.
R. Michael Roberts Dr. George A. Baumbach Philippa T. K. Saunders Thomas J. Raub Randall H. Renegar Fuller W. Bazer 《Molecular and cellular biochemistry》1986,72(1-2):67-79
Uteroferrin is a purple iron-containing acid phosphatase secreted by the porcine uterus under the influence of the hormone, progesterone. It is synthesized by the glandular epithelial cells of the uterine endometrium and during pregnancy is taken up by specialized structures (areolae) opposite each uterine gland. Uteroferrin is then released into the fetal circulation and cleared by the liver or fetal kidney. A major role in iron transport to the fetus has been proposed. Uteroferrin, as purified from uterine secretions of pigs, possesses mainly high mannose (predominately Man5 and Man6 chains. These oligosaccharide chains of uteroferrin appear to be responsible for its binding and uptake by reticuloendothelial cells of the fetal liver which is the major site of erythropoiesis of the fetus. Uteroferrin, although implicated in transplantal iron transport, also possesses many of the properties of a lysosomal enzyme and, when newly synthesized, carries the so-called lysosomal recognition marker, mannose 6-phosphate. The phosphate group is masked by a covering N-acetylglucosamine residue, a feature which may account for its secretion rather than retention within lysosomes. Evidence is also presented that the oligosaccharide chains of newly synthesized uteroferrin are larger than those of the mature form and are trimmed after secretion. The phosphate group is also removed. It is not clear whether uteroferrin carbohydrate is implicated in the movement of the glycoprotein across the placenta as well as its uptake by the fetal liver. 相似文献
17.
W R Harris D Nesset-Tollefson J Z Stenback N Mohamed-Hani 《Journal of inorganic biochemistry》1990,38(3):175-183
Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding. 相似文献
18.
Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed. 相似文献
19.
《Inorganica chimica acta》1986,125(1):1-7
Spectrophotometric work on mucic acid-W(VI) system shows the formation of three different oxoanion complexes in aqueous solution; their stability is dependent upon pH. One of the complexes is monomeric tungstodimucate and the other two are 2/2 species. An anomalous cryoscopic behaviour, similar to that of W(VI) tartaric system, has been observed for the dimeric complex formed at higher pH. The stoichiometries and conditional dissociation constants have been polarimetrically determined by means of competitive reactions between the mucic and tartaric ligands. 1H and 13C NMR spectra have been interpreted for the similar complex species of both mucic and tartaric acids. 相似文献
20.
The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H+ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H+ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10. 相似文献