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J F Hoffman

Publications and source records attributed to J F Hoffman.

At least 55 records · Page 3Linked to original sources
PubMed1979

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.

Affinity Labels↗
PubMed1979

Characteristics of anion transport in cat and dog red blood cells.

Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rats of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of 35SO4 efflux on the intracellular SO4 concentration, the pH dependence and the inhibition of 35SO4 efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of 36Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higher Vmax and K1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H2DIDS, where DIDS is 4,4'-diisothiocyano-2,2' stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H2DIDS.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1979

Heterogeneity in dog red blood cells: sodium and potassium transport.

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.

Animals↗
PubMed1979

Ionic and osmotic equilibria of human red blood cells treated with nystatin.

Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as Cl and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extracellular solution, for KCl and NaCl are 1.01 +/- 0.02 and 0.99 +/- 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]o deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, phi, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of phi Hb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH. By modifying the Jacobs-stewart equations (1947. J. Cell. Comp. Physiol. 30: 79--103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.

Body Water↗
PubMed1979

The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria.

The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

Cell Membrane Permeability↗
PubMed1979

Anion transport in dog, cat, and human red cells. Effects of varying cell volume and Donnan ratio.

Membrane potential and the rate constants for anion self-exchange in dog, cat, and human red blood cells have been shown to vary with cell volume. For dog and cat red cells, the outward rate constants for SO4 and Cl increase while the inward rate constant for SO4 decreases as cells swell or shrink. These changes coincide with the membrane potential becoming more negative as a result of changes in cell volume. Human red cells exhibit a similar change in the rate constants for SO4 and Cl efflux in response to cell swelling, but shrunken cells exhibit a decreased rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent increase in PNa. If this increase in PNa is prevented by ATP depletion or if the outward Na gradient is removed, the response to shrinking is identical to human red cells. These results suggest that the volume dependence of anion permeability may be secondary to changes in the anion equilibrium ratio which in red cells is reflected by the membrane potential. When the membrane potential and cell volume of human red cells were varied independently by a method involving pretreatment with nystatin, it was found that the rate of anion transport (for SO4 and Cl) does not vary with cell volume but rather with membrane potential (anion equilibrium ratio); that is, the rate constant for anion efflux is decreased and that for influx is increased as the membrane potential becomes more positive (internal anion concentration increases) while the opposite is true with membrane hyperpolarization (a fall in internal anion concentration).

Animals↗
PubMed1979

The Na:K pump in red cells is electrogenic.

The membrane potential, E, of the red cell measured with a fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide, hyperpolarizes when the Na:K pump is activated by adding external K and depolarizes upon the subsequent addition of ouabain. The electrogenic pump is optimally observed in cells where internal Na+ has been raised, SO2-(4) has replaced Cl-, and SO2-(4) permeability has been inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)). The change in E associated with the electrogenic component is about 6 mV in human red cells, somewhat smaller in sheep, and larger in duck and Amphiuma red cells. The membrane resistance, Rm, can be estimated from the pump-dependent change in E and from the current flow assumed to be one-third the ouabain-sensitive Na efflux. In human red cells, Rm is about 1 X 10(6) ohm-cm2. Rm calculated from the residual DIDS-insensitive SO2-(4) flux is also about 1 X 10(6) ohm-cm2. The closeness of these two values of Rm is paralleled in the other three types of red cells (even though the absolute values of Rm vary among the four types by a factor of 10), indicating that the net current flow across the membrane can be accounted for by the net transport of Na by the pump.

Animals↗
PubMed1978

Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase.

We have synthesized 2-nitro-5-azidobenzoyl (NAB) derivatives of ouabain as photoaffinity labels of the cardiac glyocoside binding site of Na, K-ATPase. [3HzNAB-ouabain was found to bind to the same number of sites on Na, K-ATPase (purified from pig kidney outer medulla) as ouabain (1.9 nmol/mg), with approximately the same affinity (Kk(ouabain)/Kd(NAB-ouabain) congruent to 1.6), and ouabain was fully competitive uith NAB-ouabain at these sites. NAB-ouabain binding and inhibition were reversible in the dark, but on exposure to ultraviolet light (310-370 nm) 30-40% of the binding and ihibition became irreversible; this binding was shown to be covalent by stability to trichloroacetic acid, organic solvents, and heat denaturation. Covalent labeling was prevented by photolysis of NAB-ouabain prior to the experiment, or by prior incubation of the enzyme with ouabain. On sodium dodecyl suffate-polyacrylamide gels of labeled Na,K-ATPase, about half of the covalently bound [3H]NAB-ouabain migrated with the large polypeptide (molecular weight congruent to 95 000), and half migrated with a small polypeptide (molecular weight congruent to 12 000); noncovalently bound NAB-ouabain (60-70% of total label) ran with the tracking dye. A similar labeling pattern was obtained utilizing NaI microsomes prepared from pig kidney outer medulla. The small polypeptide was characterized as an acidic proteolipid by extractability into acid chloroform/methanol; labeling of this component by NAB-ouabain is the first demonstration that it is directly associated with the Na,K-ATPase. The results of our characterization of NAB-ouabain show that it has the required specificity, covalency, and efficiency of labeling for application in structural studies of Na,K-ATPase subunit interactions.

Affinity Labels↗
PubMed1978

Kinetic analysis of the inhibition of sulfate transport in human red blood cells by isothiocyanates.

A kinetic analysis of anion self-exchange in human red blood cells, in the presence of an irreversible inhibitor, is presented and applied to the study of the inactivation of sulfate transport by three isothiocyanates: 3-isothiocyano-1,5-naphthalenedisulfonic acid, disodium salt (INDS), 1-isothiocyano-4-naphthalene sulfonic acid, sodium salt, monohydrate (INS), and 1-isothiocyano-4-benzenesulfonic acid, sodium salt, monohydrate (IBS). The time dependence of the inhibition of sulfate transport by the isothiocyanates used could be described by a single exponential and could be shown to contain a reversible and an irreversible component. In each case a portion of sulfate efflux was found to be resistant to inactivation. The residual portion of the sulfate efflux varied with inhibitor: 4% for INS, 16% for INDS, and 34% for IBS. INS showed the largest reversible inhibitory effect (12% of the flux remaining at 0.2 mM inhibitor concentration), while INDS showed the weakest effect (92% of the flux remaining at 0.3 mM inhibitor concentration). IBS had the highest rate of inactivation while INDS had the lowest. The kinetic analysis further suggests that all three isothiocyanates bind reversibly to an inhibitory site on the membrane before they bind covalently, and therefore irreversibly, to the same site on the membrane. The equilibrium constant for the dissociation of the reversibly-bound complex, Ki, and the rate of irreversible inactivation after all membrane sites are reversibly bound, kmax, have been computed for all three inhibitors: INDS (Ki = 420 micron, kmax = 5.04 hr-1), INS (Ki = 148 micron, kmax = 6.48 hr-1), and IBS (Ki = 208 micron, k(max) = 8.11 hr-1).

Biological Transport, Active↗
PubMed1978

Changes of intracellular Ca++ as measured by arsenazo III in relation to the K permeability of human erythrocyte ghosts.

A Ca-sensitive dye, arsenazo III, has been incorporated into resealed human erythrocyte ghosts and calibrated to monitor continuously micromolar concentrations of intracellular ionized Ca ([Ca++]i). When the external concentration of Ca is much greater than [Ca++]i, [Ca++]i increases because of a net balance between Ca influx and efflux. Dynamic changes in [Ca++]i regulate K efflux, which in turn may influence the rate of Ca influx. A procedure for purifying arsenazo III is also described.

Azo Compounds↗
PubMed1977

On the nature of the transport pathway used for Ca-dependent K movement in human red blood cells.

This paper is concerned with the mechanism by which energy-depleted human red cells become permeable to K (but not to Na) when they are exposed to Ca. In an attempt to distinguish a diffusion from a mediated process competitive type effects of different ions and their sidedness of action on K transport are considered as well as the action of certain transport inhibitors. While the nature of the interactions implies the involvement of a mediated process (perhaps an altered form of the Na:K pump apparatus) more direct evidence will be needed to make a definitive assessment.

Binding, Competitive↗
PubMed1977

Membrane compartmentalized ATP and its preferential use by the Na,K-ATPase of human red cell ghosts.

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [gamma-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.

Adenosine Triphosphatases↗
PubMed1976

Side-dependent effects of internal versus external Na and K on ouabain binding to reconstituted human red blood cell ghosts.

The side-dependent effects of internal and external Na and K on the ouabain binding rate, as promoted by inside MgATP, has been evaluated utilizing reconstituted human red blood cell ghosts. Such ghost systems provide the situation where [Na]i, [K]i, [Na]o, and [K]o can each be varied under conditions in which the others are either absent or fixed at constant concentrations. It was found that, in the presence of Ko, increasing either [Na]i or [K]i resulted in decreasing the rate at which ouabain was bound. Changes in [Na]i or [K]i in the absence of Ko were without effect on the ouabain binding rate. Thus, the ouabain binding rate was found to vary inversely with the rate of Na:K and K:K exchange but was independent of the rate of Na:Na exchange. The effect of Ko in antagonizing ouabain binding, as well as the influence of Nao on this interaction, were found to require the presence of either Nai or Ki. The results are interpreted in terms of a model relating the availability of the ouabain binding site to different conformational states of the pump complex. Differences were observed in the ouabain binding properties of red cell ghosts compared to microsomal preparations but it is not known whether the basis for the differences resides in the different preparations studied or in the lack of control of sidedness in the microsomal systems.

Adenosine Triphosphate↗