Alteration of folate analogue transport following induced maturation of HL-60 leukemia cells. Early decline in mediated influx, relationship to commitment, and functional dissociation of entry and exit routes

During treatment of HL-60 myeloid leukemia cells in culture with polar solvents or retinoic acid at a concentration inducing terminal maturation in 90-95% of the cells, there is a rapid decline (within 2 h) in the Vmax for influx of the folate analogue, [3H]methotrexate. Following 24 h of exposure to these agents, there is no effect on growth, but influx Vmax is reduced by 70%. After 7 days of exposure, influx Vmax is reduced 90-95%. A similar time course was seen for the reduction in intracellular levels of dihydrofolate reductase, a marker of cellular proliferation. Both the extent of terminal maturation (as determined by the extent of nitro blue tetrazolium reduction) and decrease in influx showed the same dependence on the concentration of inducer. In contrast to the effect seen on influx Vmax, both influx Km and mediated efflux of [3H]methotrexate remained unchanged in HL-60 cells exposed to inducers of maturation. Finally, evidence is presented for the coupling of this alteration on [3H]methotrexate influx with commitment of HL-60 cells to terminal maturation. This evidence shows that the effect on folate analogue influx precedes commitment and documents the irreversible nature of the reduction in influx once the majority of the cells exposed to inducer were committed to the process of maturation. The possible relevance of these results to the process of neoplastic transformation is discussed.     

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action.

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.     

Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity

This laboratory first provided evidence for a potential signal transduction pathway involving sphingomyelin and its derivatives (Kolesnick, R.N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). Recently, this laboratory demonstrated the existence of the novel sphingolipid ceramide 1-phosphate in human leukemia (HL-60) cells. Ceramide 1-phosphate was synthesized from ceramide derived from sphingomyelin but not glycosphingolipids. This suggested that a specific pathway extended from sphingomyelin to ceramide 1-phosphate. The present studies provide additional support for this notion by demonstrating the existence of a ceramide kinase activity distinct from diacylglycerol (DG) kinase in HL-60 cells. Microsomal membranes contained a kinase activity that phosphorylated ceramide but not 1,2-DG in the presence of physiologic and higher Ca2+ concentrations (60 nM-3 mM). Kinetic analyses demonstrated an apparent Vmax for ceramide and ATP of 70 pmol.min-1.mg protein-1; apparent Km values were 45 and 25 microM, respectively. The pH optimum was within the physiologic range (pH 6-8). Magnesium but not other divalent cations (Mn2+, Ba2+, Cd2+, Zn2+) also stimulated ceramide phosphorylation. Magnesium also induced 1,2-DG phosphorylation. Since DG kinase is a Mg2(+)-stimulable enzyme that may utilize ceramide as substrate, additional studies separated calcium-dependent ceramide kinase from DG kinase activity. 1,2-DGs competitively inhibited magnesium- but not calcium-dependent ceramide phosphorylation. Hence, calcium-dependent ceramide kinase activity neither utilized DG as substrate nor was inhibited by DG. These activities were physically separable. Both activities were solubilized by n-octyl-beta-D-glucopyranoside and stabilized by glycerol. Ceramide kinase activity bound weakly to a DEAE-cellulose anion exchange column and eluted with 4-fold purification as a single peak of activity in the flow-through and 0.05 M NaCl elutions. In contrast, the majority of DG kinase activity bound more tightly and was recovered as a broad peak in the 0.2-0.35 M NaCl elutions. These studies demonstrate the existence of a ceramide kinase activity in HL-60 cells which is functionally and physically separable from DG kinase. These studies provide further support for the notion of a specific pathway from sphingomyelin to ceramide 1-phosphate.     

Synthesis of a novel acetylated neutral lipid related to platelet-activating factor by acyl-CoA:1-O-alkyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells

Acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyl-transferase, a newly detected enzyme related to platelet-activating factor metabolism, has been characterized in microsomes of a human leukemia cell line (HL-60 cells). It has a sharp pH optimum of 6.8, does not require divalent metal ions, is stable at preincubation temperatures up to 45 degrees C, and among a variety of acyl-CoA thioesters (8:0-20:4) tested, linoleoyl-CoA is the best substrate. Km and Vmax values for 1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase are 8.5 microM and 1.7 nmol/min/mg of protein, respectively. For comparative purposes acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase was also characterized in HL-60 microsomes. It has a relatively broad pH optimum of 6.1, is stimulated 1.4-fold by Mg2+, is relatively labile at preincubation temperatures higher than 25 degrees C, and among the various acyl-CoA thioesters tested, myristoyl-CoA is the best substrate. In substrate competition experiments, we found 1-O-hexadecyl-2-oleoyl-sn-glycerol is a competitive inhibitor (Ki = 32 microM). Our findings indicate acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells is distinctly different from acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase. Our experimental results demonstrate that the unique enzyme activity characterized in this report also is expressed in intact HL-60 cells.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.     

Characterization of the Na+/H+ exchanger during maturation of HL-60 cells induced by dimethyl sulfoxide

We have studied the activity of the Na+/H+ exchanger during dimethyl sulfoxide (Me2SO)-induced maturation of the human promyelocytic leukemia cell line HL-60. 22Na uptake was measured in cells preloaded with Li+ or NH+4 in order to specifically activate the Na+/H+ exchanger. Measurement of the rate of uptake as a function of sodium concentration revealed a decrease in Km for Na+ from 38 +/- 3 to 13 +/- 1 mM after 20-24-h treatment with Me2SO. Vmax was not changed significantly. Inhibition of the exchanger by dimethylamiloride (DMA) and by acidic external pH was similar in treated and untreated cells. Thus it is unlikely that the Na+ binding site is altered. A change, however, was observed in the regulation of the exchanger by intracellular pH. In control cells maximal stimulation of the Na+ uptake was observed when the intracellular pH decreased from 7.25 to 7.00. In Me2SO-treated cells the 22Na uptake at intracellular pH 7.00 was greater than in the control and continued to increase as the intracellular pH was adjusted below 7.00, down to 6.75. This suggests that the Na+/H+ exchanger in Me2SO-treated cells is altered structurally in its allosteric H+ binding site. The appearance of this modified exchanger preceded by a period of days the appearance of a functional property characteristic of mature granulocytes, that is, the capability to produce superoxide, suggesting that the modified exchanger may be required for the expression of the mature phenotype. A second modification, a decrease in the Vmax of the 22Na uptake, occurred after 2 days treatment with Me2SO. This reduction may reflect a decrease in the number of functioning exchangers per cell.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.     

LmAPX基因在大肠杆菌和酵母菌中的表达研究

目的：研究枸杞抗坏血酸过氧化物酶基因（ascorbate peroxidase,LmAPX）在原核中的表达和酶学特性以及在酵母菌中的抗氧化能力,为进一步研究逆境诱导的抗氧化胁迫的作用机理奠定理论基础。方法：将LmAPX转入大肠杆菌BL21中进行异源表达,采用 Ni²⁺亲和层析,纯化重组蛋白,并对不同温度和pH值下的酶活进行研究,Lineweaver-Burk双倒数作图法测定该酶的Km和Vmax值。将LmAPX转入酵母菌株W303中进行H₂O₂和NaCl氧化胁迫处理。结果：该酶的最适温度和最适pH值分别为40℃和6.5。当抗坏血酸（Ascorbic acid,AsA）浓度过量时,对H₂O₂的Km和Vmax分别是0.17±0.02 mmol/L和11.78±1.88 mmol/min·mg;当H₂O₂浓度过量时,对AsA的Km和Vmax分别是2.19±0.40 mmol/L和58.82±3.51 mmol/min·mg。含有LmAPX基因的酵母菌株,在半乳糖的诱导下在8 mmol/L H₂O₂和100 mmol/L NaCl的培养基上的生长都明显优于对照组。结论：LmAPX蛋白具有很好的抗氧化性和耐盐性。     

Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons

The effect of H2O2, Cl-, and pH on human myeloperoxidase activity has been examined. The Km for H2O2 is shown to be affected by the combined presence of Cl- and acid pH conditions. The Km for H2O2 is independent of pH in the absence of Cl- and dependent on pH in the presence of Cl-. Conversely, the dependence of the Km for H2O2 on Cl- concentration increases as the pH decreases. A model is proposed in which Cl- has a dual role, acting both as a substrate and as an inhibitor. According to this model, the inhibitor Cl- binding site must be protonated prior to the binding of Cl- and is distinct from the substrate Cl- binding site which is unaffected by pH. The rate equation derived from this model is used to further analyze the data presented. The values of Km for H2O2 predicted by the rate equation are in good agreement with the experimentally determined values.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Purification and characterization of two alpha-keto ester reductases from Streptomyces thermocyaneoviolaceus IFO 14271

Two NADPH-dependent alpha-keto ester reductases (Streptomyces thermocyaneoviolaceus keto ester reductase, STKER-II and -III) were purified from S. thermocyaneoviolaceus IFO 14271, one of thermophilic actinomycetes. The molecular masses of native STKER-II and -III were estimated to be 60 kDa and 70 kDa by gel filtration chromatography, respectively. These enzymes were both homodimers, with 29-kDa and 30-kDa subunit molecular masses based on SDS polyacrylamide gel electrophoresis. STKER-II and -III were stable from pH 7.0 to 10.0 and pH 5.5 to 9.0, respectively. Ethyl 3-methyl-2-oxobutanoate was reduced by both enzymes isolated to the corresponding (R)-hydroxy ester with excellent enantiomeric excess. STKER-III showed high stereoselectivity for the reduction of bulky substrates, while the selectivity of the STKER-II-catalyzed reduction was low except for ethyl 3-methyl-2-hydrox-ybutanoate. Both enzymes had small Km values toward aliphatic keto esters having a long alkyl chain.     

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Suc uptake increased 18-fold as the pHo increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Suc uptake remained constant. The effects of pHi in the presence or absence of internal Suc were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]i) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Suc transport might be sensitive to changes in pHo. On the other hand, the [H+]i for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Suc transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Suc on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).     

Modulation of thrombin-fibrinogen interaction by specific ion effects.

Steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen, under experimental conditions where light scattering due to the formation of fibrin aggregates is negligible, have allowed for a quantitative evaluation of Km for fibrinogen. Measurements of Km for fibrinogen carried out at pH 7.5 and 37 degrees C as a function of NaCl, NaBr, KCl, and KBr concentration, from 50 to 500 mM, show that the derivative d ln Km/d ln a +/-, where a +/- is the mean ion activity, is constant over the entire range of salt concentrations and is strictly dependent on the particular salt present in solution. The values of d ln Km/d ln a +/- are found to be equal to 0.75 +/- 0.03 (NaCl), 0.90 +/- 0.01 (NaBr), 0.62 +/- 0.07 (KCl), and 0.60 +/- 0.03 (KBr). Measurements of Km for two synthetic amide substrates, under identical solution conditions, reveal practically no change in Km with salt concentration, while they show a significant decrease in kcat when Na+ salts are replaced by K+ salts. The drastic difference in the salt dependence of Km between fibrinogen and the synthetic amide substrate points out that a significant role may be played by the fibrinogen recognition site in the energetics of thrombin-fibrinogen interaction. The sensitivity of Km for fibrinogen to different salts unequivocally demonstrates that specific ion effects, rather than nonspecific ionic strength effects, modulate thrombin-fibrinogen interaction under experimental conditions of physiological relevance. Analysis of ion effects on clotting curves obtained at pH 7.5 and 37 degrees C also shows a drastic differential effect of cations and anions.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

The Na+/H+ exchanger in immature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation

We have compared the properties of the Na+/H+ exchanger in two cell populations: growing promyelocytic HL-60 cells (immature) and HL-60 cells induced to mature into granulocytes by dimethyl sulfoxide. The exchanger was activated by intracellular acidification from pH 7.25 to pH 5.5. In both immature and mature granulocytic cells, this type of activation resulted in the expected increase in Vmax for Na+ uptake but also in an increase in KmNa. Maximum acidification caused an increase in Vmax of approximately 10-fold in both types of cells. The increase in KmNa was influenced by cell maturation. In immature cells, the KmNa was higher than in mature cells at all pH values tested, and this difference increased with acidification. Maximum acidification increased the KmNa from 15 +/- 4 to 124 +/- 17 mM in immature cells and from 10 +/- 3 to 43 +/- 20 mM in mature cells. Intracellular pH also influenced the pattern of inhibition of 22Na uptake by dimethylamiloride, a specific inhibitor of the exchanger. At intracellular pH 7.0 dimethylamiloride inhibition was mostly competitive in immature and competitive in mature cells. At lower intracellular pH, 5.9, the inhibition was mixed in both types of cells. Thus, the properties of the exchanger in granulocytic cells are influenced by the cell maturation stage and the intracellular pH.