Co-operating ATP sites in the multiple drug resistance transporter Mdr1.

The ATPase activity of the multiple drug resistance transporter Mdr1 (P-glycoprotein, gp170) depended on the concentration of ATP with both positive and negative co-operativity both in the absence and in the presence of verapamil. Four co-operating binding sites for ATP were required to adequately model the experimental findings. The activation energy for the ATPase activity increased from approximately 385 kJ x mol-1 at 10 microM ATP to 512 kJ x mol-1 at 1600 microM, while changes in verapamil concentration had little effect. This indicates that the reaction mechanism of ATP hydrolysis depends on ATP concentration and is further evidence for co-operation of ATP binding sites. Free ATP in higher concentration was inhibitory; however, this inhibition could be reduced by complexing the ATP with Mg2+. Free Mg2+ had little effect on Mdr1 apart from complexing ATP.

Co-operative binding sites for transported substrates in the multiple drug resistance transporter Mdr1.

Suramin, a known inhibitor of ATP binding enzymes with six negatively charged sulfonic acid groups, stimulated the ATPase activity of the multiple drug resistance transporter Mdr1 in low concentrations by acting as a substrate and by increasing the affinity for both verapamil and ATP. At higher concentrations suramin inhibited the ATPase activity competitively with respect to ATP and noncompetitively with respect to verapamil. This indicates an interaction of suramin with the ATP site. Verapamil itself activated the ATPase activity of Mdr1 only at moderate concentrations, but showed substrate inhibition at higher concentrations. This was also observed for progesterone, which decreased the Ki of Mdr1 for verapamil but increased the Km. Additionally, verapamil increased the Hill coefficient of Mdr1 for progesterone from 1.1 to 3.2. These results indicate the existence of multiple binding sites (at least four for progesterone) for transported substrate in Mdr1 and a complicated mode of interactions between them.

Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein).

Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1 +/- 0.1 microM and 1.4 +/- 0.7 mM respectively at 25 degrees C. This corresponds to delta G0' = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, delta H0' was calculated to -36 +/- 2 kJ/mol. This gives delta S0' = 6.7 J/mol per K. Adenosine 5'-[gamma-thio]triphosphate, ADP, adenosine 5'-[beta, gamma-imino]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 microM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.

The speed of partial reactions of the uncoating ATPase Hsc70 depends on the source of coated vesicles.

Hsc70 was previously isolated by its ability to catalyse the uncoating of clathrin-coated vesicles from bovine brain. We have recently shown that Hsc70 is more active towards coated vesicles from brain than those from other tissues. In order to gain information on the mechanistic reason for this difference we have examined the ability of brain and placental coated vesicles to stimulate partial reactions during a single round of ATP turnover. The Hsc70-ATP complex is turned over to Hsc70-ADP center dot Pi, from which phosphate is slowly released. The resulting Hsc70-ADP complex exchanges ATP for ADP. Dissociation of ATP or ADP from Hsc70 does not seem to occur under physiological conditions. The hydrolysis of ATP is accelerated by the presence of clathrin-coated vesicles, with vesicles from brain being about twice as effective as vesicles from placenta. Additionally, it appears that brain, but not placental, coated vesicles can also stimulate the exchange of ADP for ATP.

Selective action of uncoating ATPase towards clathrin-coated vesicles from brain.

Clathrin-coated vesicles from brain are primarily involved in synaptic vesicle recycling and are substrates for the constitutively expressed heat shock cognate hsc70 protein (uncoating ATPase). To investigate the regulation of clathrin coat turnover in other tissues the activity of hsc70 towards coated vesicles from other sources was examined. Concentrations of hsc70 which caused near-complete removal of clathrin from brain coated vesicles effected only partial uncoating of vesicles prepared from other tissues. The selective action of hsc70 could not be accounted for by tissue or species specificities of hsc70, but rather reflected differences in coat structure. Selective action was associated with two differences in the hsc70-dependent ATPase cycle. Firstly, uncoating of brain, but not placental vesicles, could occur under circumstances where ATP hydrolysis was prevented. Secondly, only brain coated vesicles could support multiple rounds of hsc70-dependent ATP hydrolysis. Implications of these findings for the mechanism of hsc70-dependent vesicle uncoating in non-neuronal cells and the organisation of the endocytic pathway in the axon are discussed.

Construction and use of two alpha-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype alpha-hANP biosensor.

Two alpha-human atrial natriuretic peptide (alpha-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of alpha-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate alpha-hANP-specific antibodies from a goat anti-alpha-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an alpha-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological alpha-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This alpha-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.

Investigation of subunit interactions by radiation inactivation: the case of Na+/K+-ATPase.

The target size of Na+/K+-ATPase has been determined by radiation inactivation. To interpret the results, we have performed Monte Carlo simulations of the inactivation process. This seems to be a general method for the interpretation of such studies. The simulation revealed that radiation inactivation can distinguish between monoprotomeric and multiprotomeric models of enzyme action only if the measured reaction requires the actual co-operation of, rather than the mere co-existence of, different protomeres.

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na(+)-exchange reaction of the sodium pump unaltered and shifts its high-affinity ATP-binding site to a Na(+)-like form.

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.

Quantitative determination of pectic substances as an example of a rhamnopolysaccharide assay.

A quantitative enzymatic assay for rhamnopolysaccharides is described. The procedure is shown with pectic substances as an example. The test is based on the enzymatic degradation of the macromolecules to liberate L-rhamnose. This sugar can be quantitatively determined with the help of L-rhamnose dehydrogenase under concomitant reduction of NAD, thus allowing the quantitative evaluation of the original pectin.