PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria: The role of potassium

In previous studies it was found that change in the concentrations of Ca²⁺, H⁺, and HPO₄²⁻ in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K⁺ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)₂D-3 synthesis by isolated chick mitochondria; that the magnitude of this K⁺-dependent stimulation is enhanced by optimal concentrations of calcium (pCa = 5) and phosphate (pP_i = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na⁺ concentration; and that the stimulatory effect of K⁺ is not blocked by ruthenium red, an inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH)₂D-3 synthesis. It was also found taht valinomycin, a K⁺-specific ionophore, enhanced the sensitivity of the mitochondrial 1α-hydroxylase activity to K⁺. In the presence of valinomycin, an increase of pK⁺ to 3 was sufficient to cause a significant stimulation of 1,25(OH)₂D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulate the activity of the 1α-hydroxylase.     

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA

The interaction of the Cu(II) drugs CuL(NO₃) and CuL′(NO₃) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL′ is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL]⁺ and [CuL′]⁺) with [poly(dA–dT)]₂, [poly(dG–dC)]₂, and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 °C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA–dT)]₂, with a site size n = 6–7, whereas they act as external binders with [poly(dG–dC)]₂ and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL′]⁺ were slightly larger than with [CuL]⁺. The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL]⁺ and [CuL′]⁺, with a significative dose–response effect for [CuL′]⁺, which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH₂ group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.     

Identification of a rabbit B cell alloantigen with an antiserum produced by alloimmunization with thymus cells.

Alloimmunization with rabbit thymus cells resulted in an antiserum (anti-Rly) which was shown to react with rabbit lymphocytes by an indirect rosette technique. The titration curve obtained with dilutions of anti-Rly antiserum on lymph node cells revealed two plateaus indicating that the antiserum was multispecific; at low dilutions of antiserum, within the first plateau, both B and T cells were rosetted whereas at high dilutions, within the second plateau, only B cells were rosetted. The antigen detected at high dilution was designated LB-1 (lymphocyte B cell alloantigen 1). The evidence that the cells identified within the second plateau are B cells is as follows: 1) simultaneous enumeration of LB-1⁺ and Ig⁺ (B) cells by use of distinguishable erythrocytes (sheep and human) as indicator cells revealed that of the 53% rosettes observed, essentially all (51%) were mixed rosettes containing both erythrocytes whereas simultaneous enumeration of LB-1⁺ and T⁺ cells (identified by anti-T cell antiserum) showed essentially no mixed rosettes (less than 2%); 2) approximately 80% of purified Ig⁺ (B) cells were identified as LB-1⁺ cells whereas essentially no (< 1%) purified T cells could be detected as LB-1⁺; 3) the percentages of LB-1⁺ cells and Ig⁺ cells were both reciprocal to the precentages of T⁺ cells identified in various lymphoid organs except for bone marrow; 4) the removal of LB-1⁺ cells from spleen cells of rabbits immunized with sheep red blood cells resulted in a depletion (42–71%) of direct plaque forming cells (PFC). Since the percentages of bone marrow cells rosetted using anti-LB-1 antiserum (approximately 70%) was much greater than the percentage rosetted using anti-Ig (approximately 10%), it appears that the anti-LB-1 antiserum is not directed against an Ig allotype. The titration curves of the anti-Rly antiserum on peripheral blood lymphocytes of a large rabbit family suggested that the LB-1 antigen on B cells is an alloantigen probably inherited in simple Mendelian fashion. Adsorption studies indicated that the LB-1 antigen on B cells is not detectable on brain, liver, kidney or erythrocytes.     

Mechanism of aldosterone-induced increase of K+ conductance in early distal renal tubule cells of the frog

Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K⁺ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K⁺ conductance (gK⁺) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK⁺ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH _i ) could be a signal for cells to regulategK⁺. After removal of aldosterone, the increase ingK⁺ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH _i and doubledgK⁺.Using the patch-clamp technique, three types of K⁺-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P _o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P _o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH _i and increasesgK⁺ by promoting insertion of K⁺ channels into the cell membrane. Channel insertion is itself triggered by raising both pH _i and increasing the activity of the Na⁺/K⁺ pump in early distal cells of frog kidney. Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England     

Deoxyribokinase from Salmonella typhimurium. Purification and properties

Deoxyribokinase, which catalyzes the ATP-dependent phosphorylation of 2-deoxy-d-ribose to 2-deoxy-d-ribose-5-P as the first step in the inducible fermentation pathway for this sugar in Salmonella typhimurium, was purified approximately 600-fold from deoxyribose-grown cells. Apparent K_m′s for 2-deoxy-d-ribose and ATP were 0.1 and 0.5 mm, respectively. The enzyme had an absolute requirement for divalent cations which was best satisfied by Mg²⁺. Optimal activity was obtained in the presence of 0.5 m NH₄⁺ or Cs⁺. Rb⁺ and K⁺ also stimulated enzyme activity whereas Na⁺ and Li⁺ inhibited. d-Ribose and 2-deoxy-d-ribitol could replace 2-deoxy-d-ribose as phosphoryl acceptor, and several ribo- and deoxyribonucleotides could replace ATP as phosphoryl donor. Molecular weight determinations gave values of 67,800 for the native enzyme and 33,500 for the dissociated enzyme, suggesting the probable existence of two subunits of similar size.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.     

Hexokinases of spinach leaves

Two soluble hexokinases and a particulate hexokinase have been separated and partially purified from spinach leaves. One of the soluble hexokinases showed a high affinity for glucose (K_m = 63 μM) which was far greater than that for fructose (K_m = 9.1 mM). However, with saturating fructose the activity was twice that with saturating glucose. The particulate hexokinase showed kinetic properties similar to those of this soluble hexokinase. The second soluble hexokinase was distinct in that it was much more active with fructose than with glucose at all concentrations tested, although the K_m values for these hexoses (210 μM and 71 μM respectively) were similar. The activity of this hexokinase was stimulated by the monovalent cations K⁺ and NH₄⁺.     

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.     

Equilibrium and structural studies of silicon(IV) and aluminium(III) in aqueous solution. 12. A potentiometric and 29Si-NMR study of silicon tropolonates

Complexation in the H⁺-Si(OH)₄-tropolone (HL) system was studied in 0.6 M (Na)Cl medium at 25° C. Speciation and formation constants were determined from potentiometric (glass electrode) and ²⁹Si-NMR data. Experimental data cover the ranges 1.5 ? - log[H⁺] ? 8.4, 0.002 ? B ? 0.012 M, and 0 ? C ? 0.060 M (B and C stand for the total concentration of Si and tropolone, respectively). In acid solutions (-log[H⁺] ? 3) a hexacoordinated cationic complex, SiL₃⁺, is formed with log K(Si(OH)₄ + 3HL + H⁺ XXX SiL₃⁺ + 4H₂O) = 7.08 ± 0.03. Furthermore, the formation of a disilicic acid was established from ²⁹Si-NMR data. The dimerization constant of Si(OH)₄ was found to be 10 exp (1.2 ± 0.1). In model calculations the solubility of quartz and amorphous SiO₂ in the presence of tropolone is demonstrated. Data were analyzed using the least-squares computer program LETAGROPVRID.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

Effect of fluorine position on the coordinating ability of fluorosalicylic acids--an experimental study complemented with computations

The complexation of 3-, 4-, and 6-fluorosalicylic acids (HL) with copper(II) was investigated in aqueous solution by pH-potentiometry combined with UV-visible spectrophotometry, and in 50 v/v % water-methanol mixture by the two-dimensional ESR simulation method. Both methods showed the formation of [CuLH₋₁] and [CuL₂H₋₂]²⁻ of high stabilities, and, at low excess of ligand, the ESR-silent mixed hydroxido complex [Cu₂L₂H₋₃]⁻. Further species were also identified by the two-dimensional ESR simulation method: [CuL]⁺ in the acidic region, the minor dimer [Cu₂L₂H₋₂], and the cis and the trans isomers for [CuL₂H₋₂]²⁻. The position of the fluorine atom in the aromatic ring had significant effect on the coordination abilities of the ligands, in good correlation with their reported biological activities. It was 3-fluorosalicylic acid, which formed the most stable complexes [CuLH₋₁] and [CuL₂H₋₂]²⁻, while the mononuclear complexes with 6-fluorosalicylic acid were found to be the least stable. For the other ligands (including 5-fluorosalicylic acid studied recently), complexes of medium stabilities were formed. For the interpretation of these findings, ab initio and semi-empirical quantum chemical calculations were carried out for the ligand molecules, isolated and surrounded by water molecules, respectively.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Sodium-dependent transport of 5-hydroxytryptamine (serotonin) by canine blood platelets

The transport of 5-hydroxytryptamine (5-HT) was shown to be strongly dependent on the presence of Na⁺ in the incubation medium whereas divalent cations were without effect. The K_m for the Na⁺ requirement was 16.8 mm. The addition of Na⁺ to Na⁺-depleted platelets restored maximum 5-HT transport within 3 min. The affinity of the 5-HT carrier for its substrate was directly proportional to the concentration of Na⁺; however, below 25 mm Na⁺ unique reversible morphological changes in platelet shape occurred as revealed by scanning electron microscopy which resulted in a drastically reduced affinity for 5-HT. K⁺, choline (Ch⁺), or Li⁺ could be used as counterbalancing cations to maintain osmolarity, and the affinity for 5-HT was also dependent on the concentrations of these ions. Ouabain as well as various ionophores at low concentrations inhibited 5-HT uptake. The inhibition was the result of the destruction of the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ gradient across the cytoplasmic membrane. Ionophores, however, did not cause the depletion of either intracellular ATP or 5-HT.     

Inhibition of red cell and yeast hexokinase by triethyltin bromide [(C2H5)3SnBr]

Triethyltin bromide was found to be a selective inhibitor of red cell hexokinase. When intact red cell suspensions were exposed to the reagent (0.025–0.5 mM), inhibition of hexokinase occurred without any hemolysis and without effects on the activity of other glycolytic enzymes. Yeast hexokinase was also inhibited by triethyltin. The red cell and yeast hexokinases were protected against inhibition by triethyltin when the sugar substrates of the enzymes were included in the incubation mixture. This work identifies hexokinase as another among a limited number of proteins that are known to interact with triethyltin.     

Inhibition of K+-sensitive p-nitrophenylphosphatase activity on rhesus-positive red cells after incubation with IgG-anti-rhesus-D

The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K⁺-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na⁺ + K⁺)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K⁺-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl₃ at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells.