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The separation of proteins by gel-exclusion chromatography has been explained in terms of partitioning of the macromolecules within the gel by a distribution of pores of various radii. The assumption that the distribution of pore sizes is Gaussian has led to the prediction of a linear relationship between the molecular Stokes radius (RS) of the protein and the function erf-1 (1-KD), where KD is the partition coefficient [Ackers (1967) J. Biol. Chem. 242, 3237-3238]. Since careful calibrations of classical (agarose and dextran) gels and h.p.l.c. gels have shown that such a linear relationship is not verified experimentally over a wide range of native protein sizes, we have reinvestigated the model of Ackers (above reference). We show that Ackers' (above reference) derivation is not valid except for a particular Gaussian distribution of pore sizes centred at the origin. Relaxation of this restriction to allow for other types of Gaussian distributions cannot account for the non-linear calibration curves that we have obtained. Instead we show that the pore-size distribution can be calculated from the experimentally determined function KD = f(RS) and that this distribution is bimodal (non-Gaussian). One distribution is centred below 2 nm, whereas the mean value of the second one is around 6-8 nm. The minimum in this bimodal distribution corresponds, for some gels, to a region of poor resolution, which needs to be appreciated for the proper use of gel chromatography in the determination of molecular size.

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.

1. The renal excretion of phenol red and other phenolsulphophthalein dyes (bromophenol blue and bromothymol blue) was studied in clearance experiments on anaesthetized rabbits. 2. Net tubular excretion of phenol red reached a maximal value of 8 mumole/min at a plasma concentration of ultrafiltrable dye of about 0.1 mM and was decreased at higher plasma concentrations. Decreases in net tubular excretion at high plasma concentrations were also obtained for bromophenol blue and bromothymol blue suggesting tubular reabsorption in addition to tubular secretion of the dye. Conclusive evidence for reabsorption was provided by administration of probenecid which caused a fall in the excretion of the dyes below that filtered by the glomeruli. 3. Tubular reabsorption of phenol red during probenecid administration appeared to be proportional to the glomerular load and was increased under experimental conditions leading to a decrease of urinary pH. Experiments involving efflux of phenol red from liposomes gave no evidence of a significant role of transmembrane passage by non‐ionic diffusion. It is suggested that the pH dependence of the reabsorptive process is the result of preferential reabsorption of the acid as compared to the basic form of the indicator dye across a hydrophilic pathway in the transporting membranes. 4. Clearance ratio of phenol red to that of p‐aminohippurate at low plasma concentrations was about 0.3. They low degree of extraction of phenol red from renal plasma is attributed both to tubular reabsorption and binding of the dye by plasma proteins.

1. The relation between the coupling of metabolic energy to renalp‐aminohippurate (PAH) accumulation and Na+‐K+ transport was studiedin rabbit cortical slices. 2. Cyanide (CN‐), 2,4‐dinitrophenol (DNP)and fluoride (F‐( at low‐medium concentrations, giving rise to aslight decline of tissue ATP concentration, caused a reduction o PAHaccumulation without significantly affecting intracellular Na+ and K+concentrations. However, higher levels of the metabolic inhibitorsalso resulted in considerable inhibition of active Na+‐K+transport. 3. The rate of carrier‐mediated PAH uptake was slow underanaerobic conditions, relative to that measured under aerobicconditions in Na+‐depleted slices. In the latter case the maximalaccumulation achieved was only 1.55 +/‐ 0.16. 4. The uptake rate ofPAH under anaerobic conditions was not inhibited by the absence of Na+or addition of metabolic inhibitors in the concentrations used underaerobic conditions. 5. It is concluded that although Na+ is requiredfor the attainment of high accumulation ratios of PAH, oxidativemetabolism stimulates PAH flux by a Na+‐independentmechanism.

1. The relation between renal accumulation of p‐aminohippurate (PAH), Na+‐K+‐ATPase activity, and the transmembranal Na+ gradient has been investigated by the use of cortex slices of rabbit kidney. 2. A moderate stimulation of PAH uptake rate was observed under anaerobic conditions in the presence of an extracellular‐to‐intracellular directed gradient of Na+. 3. Inhibition of aerobic accumulation of PAH by ouabain was related to a decrease in Na+‐K+‐ATPase activity, as evidenced by changes in tissue concentrations of Na+ and K+ and by measurements of high‐affinity binding of ouabain to the slices. 4. Accumulation of PAH in media with various concentrations of Na+ and K+ resembled the effect of the cations on the ATPase activity of an isolated preparation of Na+‐K+‐ATPase. 5. Counteraction of the inhibitory effect of ouabain on PAH accumulation by a high concentration of K+ in the medium was related to retention of Na+‐K+‐ATPase activity, as evidenced by preservation of tissue‐‐medium gradients of Na+ and K+. 6. The present data provide strong evidence for the involvement of Na+‐K+‐ATPase in the energization of renal PAH accumulation. However, it appears probable that a metabolic (Na+‐gradient‐independent) component, in addition to a Na+ gradient, is essential for the attainment of high accumulation ratios of PAH by intact renal cells.

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P—-E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.

The properties of detergents required to substitute the lipid environment of sarcoplasmic reticulum Ca2+-ATPase with retention of good functional properties were determined by the use of a large number of diverse detergents and delipidated enzyme. Detergents having an intermediate chain length (≈C12) and a polyoxyethylene glycol or carbohydrate polar group were optimal for Ca2+-ATPase function and stabilization, while detergents with short alkyl chain (C8) or bulky head groups and many zwitterionic detergents led to rapid inactivation. Under optimal conditions (including solubilization in the E1state), stability of delipidated Ca2+-ATPase approximated that obtained by solubilization of Ca2+-ATPase with a layer of bound lipid. Some detergents (in particular long chain members of the Tween family) were characterized by an inadequate interaction with delipidated Ca2+-ATPase, resulting in biphasic inactivation. According to analytical ultracentrifugation and high performance liquid chromatography experiments, the rapid and slow components of biphasic inactivation were due to the formation of monomeric and oligomeric Ca2+-ATPase, respectively.

Changes in Ca2+ binding after phosphorylation of membranous or detergent-solubilized preparations of sarcoplasmic reticulum Ca2+-ATPase with ATP were followed spectrophotometrically by the use of murexide. Distinct Ca2+ release from the two high-affinity translocation sites was observed, particularly at alkaline pH and at low Ca2+/Mg2+ concentration ratios. Phosphorylation also induced additional binding of Ca2+ at a third site in competition with Mg2+. Ca2+ release was increased after solubilization of Ca2+-ATPase in predominantly monomeric form with the nonionic detergent octaethyleneglycol monododecyl ether. At 0 degree C, chemical-quench studies with [32P]ATP indicated that release of Ca2+ is correlated with the level of ADP-insensitive phosphoenzyme (2 mol of Ca2+ released per mol of E2P formed), both for membranous and detergent solubilized Ca2+-ATPase. Ca2+ release was also found to be accompanied by changes in intrinsic fluorescence. Analysis of the data at 20 degrees C, pH 8.0, showed that binding of Ca2+ to transport sites on E2P occurs with a half-saturation constant of 0.7 mM and a Hill coefficient of 1.8. This is consistent with a drastic decrease in Ca2+ affinity following conversion of ADP-sensitive E1P to ADP-insensitive E2P. The similarity between membranous and detergent-solubilized Ca2+-ATPase supports the view that not more than a single Ca2+-ATPase polypeptide chain is required to complete the conformational transitions which are the basis for active transport of Ca2+.

Transport of rho-aminohippurate was studied by the use of a preparation of rabbit kidney basolateral-membrane vesicles and in rat kidney-cortex slices under anaerobic conditions. With both preparations clear evidence of Na+-gradient stimulation of rho-aminohippurate transport (‘overshoot’) was obtained. These results thus indicate that a significant aspect of active rho-aminohippurate transport is by co-transport with Na+, and they appear to resolve previous disagreements concerning the role of Na+. Vesicle studies with a potential-sensitive dye suggested that rho-aminohippurate may be transported electroneutrally, i.e. in a 1:1 complex with Na+.

The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypeptide chain is the functional unit in energy transduction.

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.

1. The interaction of the haem-binding region of apomyoglobin with different ligands was examined by ultrafiltration, equilibrium dialysis and spectrophotometry, to study unspecific features of protein-ligand interactions such as they occur in, for example, serum albumin binding. 2. Apomyoglobin, in contrast with metmyoglobin, binds at pH 7, with a high affinity, one molecule of Bromophenol Blue, bilirubin and protoporphyrin IX, two molecules of n-dodecanoate and n-decyl sulphate and four molecules of n-dodecyl sulphate and n-tetradecyl sulphate. 3. The number of high-affinity sites and/or association constants for the alkyl sulphates are enhanced by an increase of hydrocarbon length, indicating hydrophobic interactions with the protein. 4. Measurements of the temperature-dependence of the association constants of the high-affinity sites imply that the binding processes are largely entropy-driven. 5. Binding studies in the presence of two ligands show that bilirubin plus Bromophenol Blue and dodecanoate plus Bromophenol Blue can be simultaneously bound by apomyoglobin, but with decreased affinities. By contrast, the apomyoglobin-protoporphyrin IX complex does not react with Bromophenol Blue. 6. Optical-rotatory-dispersion measurements show that the laevorotation of apomyoglobin is increased towards that of metmyglobin in the presence of haemin and protoporphyrin IX. Small changes in the optical-rotatory-dispersion spectrum of apomyoglobin are observed in the presence of the other ligands. 7. It is concluded that the binding sites on apomyoglobin probably do not pre-exist but appear to be moulded from predominantly non-polar amino acid residues by reaction with hydrophobic ligands. 8. Comparison with data in the literature indicates that apomyoglobin on a weight basis has a larger hydrophobic area avaialble for binding of ligands than has human serum albumin. On the other hand, the association constants of serum for the ligands used in this study are generally somewhat larger than those of apomyoglobin.