The isoelectric point, a key to understanding a variety of biochemical problems: a minireview.

We address the importance of the isoelectric point (IEP) of proteins and membrane components such as phospholipids for our understanding and interpretation of isoforms and opposite charge interactions in the formation of complexes. Five examples drawn from the literature are newly approached from the IEP point of view to clarify general principles.

Organ-related distribution of phospholemman in the spiny dogfish Squalus acanthias.

The distribution of phospholemman among nine different organs of the spiny dogfish (Squalus acanthias) has been determined on the basis of Western blotting of microsomal material. Only rectal gland (100%), brain (43%), heart (18%), and kidney (19%) (abundancies as percent of the concentration in rectal gland) contained the protein, but not gill and colon. The relative abundance in the brain makes this organ a preferential test system for phospholemman in fishes that lack a rectal gland like teleosts.

N-terminal sequences of small ion channels in rectal glands of sharks: a biochemical hallmark for classification and phylogeny?

The rectal gland of sharks contains a 13.2-kDa microsomal protein that in primary structure resembles to a variable extent the mammalian Cl- channel phospholemman. It appears to reside in basolateral as well as in apical membranes. The large variation in primary structure among different orders and families of sharks could make the protein a hallmark for classification.

Constitutive activation of gastric H+,K+-ATPase by a single mutation.

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.

E31-K352, the minimal cation binding moiety of Na+,K(+)-ATPase.

Upon limited tryptic fragmentation of Na+,K(+)-ATPase a 35 kDa fragment (E31-K352) was formed that bound 204Tl+ on blot. Further fragmentation led to loss of binding, pointing to the conclusion that E31-K352 is the minimal cation binding unit in Na+,K(+)-ATPase.

Kinetics of Cu2+ inhibition of Na+/K(+)-ATPase.

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mixed-type inhibitory effect with respect to Mg2+. It is concluded that the appreciation of the inhibitory effect of Cu2+ on this enzyme requires carefully composed assay media that include a buffer for Cu2+, and that the IC50-values calculated according to this model indicate that Cu2+ may be more toxic than previously anticipated.

Sodium acts as a potassium analog on gastric H,K-ATPase.

The effects of Na+ on gastric H,K-ATPase were investigated using leaky and ion-tight H,K-ATPase vesicles. Na+ activated the total ATPase activity in the absence of K+, reaching levels of 15% relative to those in the presence of K+. The Na+ activation, which takes place at the luminal side of the membrane, depended on the ATP concentration and the type of buffer used. The steady-state ATP phosphorylation level, studied with leaky vesicles, was reduced by Na+ due to both activation of the dephosphorylation reaction and a shift to E2 in the E1<==>E2 equilibrium. By studying this equilibrium in ion-tight H,K-ATPase vesicles, it was found that Na+ drives the enzyme via a cytosolic site to the nonphosphorylating E2 conformation. No H(+)-like properties of cytosolic Na+ could be detected. We therefore conclude that Na+ behaves like K+ rather than like H+ in the H,K-ATPase reaction.

Tertiary amines as antagonists of both the luminal and cytosolic K(+)-site of gastric H,K-ATPase.

Tertiary amines like imidazole and triallylamine lower the apparent affinity of K+ in the ATP hydrolysis reaction of pig gastric H,K-ATPase in a pH and amine concentration dependent way. The mechanism and sidedness of this effect was studied by analyzing the partial reactions of the enzyme in both leaky and ion-tight vesicles. In leaky vesicles Tris and Hepes had nearly no effect on the apparent Km for K+ in the ATPase reaction, but imidazole (Ki = 13 mM) and triallylamine (Ki = 1.6 mM) markedly decreased the K+ affinity. The steady-state ATP-phosphorylation level in the absence of K+ was not or only slightly affected by these compounds. The reduction of the ATP-phosphorylation level by K+, however, again depended on both the type and concentration of tertiary amine used. A comparable K(+)-amine antagonism was observed in the dephosphorylation reaction. In tightly sealed vesicles, where no activation of K+ at the luminal side could occur, K+ reduced the affinity for ATP in the phosphorylation reaction. Triallylamine counteracted this effect. The K(+)-activated p-nitrophenylphosphatase activity in these ion-tight vesicles also showed a K(+)-triallylamine antagonism. Inhibition of H,K-ATPase activity in these vesicles by triallylamine was immediate (with nigericin present in order to allow intravesicular K+ activation), suggesting the transmembrane feature of this inhibition. These results indicate that tertiary amines decrease the affinity for K+ at both luminal and cytosolic binding sites by interaction at the cytosolic side of the membrane. This results in shifts in the equilibrium of both the E1.H<==>E1.K transition and in the dephosphorylation reaction, E2-P-->E2.K.

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.

Binding of unsaturated fatty acids to Na+, K(+)-ATPase leading to inhibition and inactivation.

The effects of free fatty acids on the mechanism of action of Na+, K(+)-ATPase were studied. Unsaturated free fatty acids (palmitoleic acid, oleic acid, linoleic acid and arachidonic acid) inhibited the Na+, K(+)-ATPase activity within a narrow range, while saturated and methylated fatty acids had little or no effect. The following effects of oleic acid were found: (1) The affinity for K+ on the overall ATPase and the p-nitrophenylphosphatase reaction as well as the maximal activities were decreased. (2) The Na(+)-ATPase activity was also inhibited but the '0'-ATPase activity was hardly changed. (3) The steady-state ATP phosphorylation level in the presence of Na+ was not influenced. (4) The dephosphorylation rate constant of the phosphointermediate was slightly decreased, resulting in elevated phosphorylation levels in the absence of Na+. (5) The inhibitory effect of ATP on the dephosphorylation rate was not affected. (6) The K+ sensitivity of the phosphoenzyme in the presence as well as in the absence of Na+ was decreased. (7) Ouabain binding was inhibited. Both the affinity and the number of binding sites were lowered. In addition it was found that Na+, K(+)-ATPase binds oleic acid linearly with the fatty acid concentration up to more than 100 mol oleic acid per mol alpha beta oligomer of Na+, K(+)-ATPase. Prolonged incubation with oleic acid led to irreversible inactivation of the enzyme. This inactivation was dependent on the reaction conditions: ligands, temperature, enzyme concentration, time and fatty acid concentration. The combined presence of inactivation (long term effects) and the effects on the (K(+)-activated) dephosphorylation (short term effects) explain the mixed type inhibition of free fatty acids as observed in assays for K(+)-activated ATPase, K(+)-activated p-nitrophenylphosphatase and ouabain binding. It also explains the sharp inhibition curve in the Na+, K(+)-ATPase activity test.

Sidedness of the effect of amines on the steady-state phosphorylation level of reconstituted Na+/K+-ATPase.

The sidedness of the effects of several amines on the steady-state phosphorylation level of rabbit kidney Na+/K+-ATPase has been studied with the enzyme incorporated in phosphatidylcholine-cholesterol containing proteoliposomes. The presence of ouabain prevented phosphorylation of non-incorporated or rightside-out incorporated enzyme, so that only the inside-out incorporated Na+/K+-ATPase molecules were studied. Addition of either Na+ or several amines to the extracellular side of the enzyme led to an enhancement of the steady-state phosphorylation level obtained with optimal concentrations of Na+, Mg2+ and ATP at the cytosolic side. The series imidazole greater than Na+ greater than triallylamine greater than Tris greater than ethylenediamine showed a decrease in affinity. Histidine, sorbitol and choline chloride had no effect at the extracellular side. This means that in addition to the well-known cytosolic ligands either Na+ or a positively charged amine buffer has to be present extracellularly in order to obtain an optimal phosphorylation level. At the cytoplasmic side the tested amines exerted different effects. (i) Imidazole and triallylamine enhanced the steady-state phosphorylation level when the extracellular conditions were optimal (saturating amine concentration). (ii) Tris and ethylenediamine decreased the steady-state phosphorylation level and (iii) histidine had no effect. The cytoplasmic effects of the amine compounds correlate with those described by Schuurmans Stekhoven et al. (Biochim. Biophys. Acta 937 (1988) 161-171) for the unsided preparation. The extracellular effects, however, are apparently masked in experiments with fragmented enzyme preparations and are assumed to be potentiating effects which make the enzyme ready for phosphorylation upon a cytoplasmic trigger (e.g. Na+).

Ethylenediamine as active site probe for Na+/K+-ATPase.

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.

Phosphorylation of (Na+ + K+)-ATPase; stimulation and inhibition by substituted and unsubstituted amines.

(1) In view of a previously established stimulation of steady-state phosphorylation of (Na+ + K+)-ATPase by imidazole and its inhibition by tris(hydroxymethyl)aminomethane, the effect of (structure, chemical composition and charge of) a number of primary, secondary and tertiary amines (including imidazole derivatives) has been investigated. (2) Primary amines are predominantly inhibitory and diamines are more inhibitory than monoamines. The strongest inhibition is exerted by ethylenediamine (I50 in 50 mM imidazole = 25 microM, vs. 60 mM for n-propylamine). Increasing the distance between the two amino groups from 3.7 to 8.7 A increases the I50 180-fold. The optimal distance of 3-4 A indicates a similar distance between two ligand(presumably Na+)-binding sites on the enzyme. (3) Screening or substitution of the central N-atom decreases inhibition by the nitrogen compound. Triple substitution by propyl or allyl groups leads to maximal activation, amounting to about 90% of the Na+-activation level. Triethyl substitution gives suboptimal activation and tributyl substitution leads to inhibition. Substitution by polar or negatively charged carboxyl groups diminishes or even abolishes inhibition and also diminishes or abolishes activation. (4) Although occasionally positive charge is not required for inhibition, it is prerequisite for activation. Within certain families of compounds (e.g., ethylenediamine and imidazole derivatives) inhibition or activation increases with pKa, hence with positive charge. (5) The above data are interpreted in terms of inhibition, which is competitive to Na+, being governed by Coulomb interaction. Activation, on the other hand, is predominantly determined by lipophilic (van der Waals or pi-pi electron) interactions, excluding water from the phosphorylation site, hence decreasing phosphoenzyme hydrolysis and increasing the phosphoenzyme level. The requirement of charge (though hidden by substitution) implies weak additional electrostatic interaction.

Pb2+ and imidazole-activated phosphorylation by ATP of (Na+ + K+)-ATPase.

In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+.

Nucleotide specificity of the E2K----E1K transition in (Na+ + K+)-ATPase as probed with tryptic inactivation and fragmentation.

The nucleotide specificity for the E2K----E1K conformational transition in (Na+ + K+)-ATPase as the key step for overall hydrolytic activity and coupled cation transport has been investigated. Use has been made of tryptic inactivation, which is biexponential in time for the enzyme in the presence of Na+ with or without nucleotides (E1 conformation) and monoexponential in the presence of K+ (E2 conformation). ATP, AdoPP[NH]P and CTP in order of decreasing effectivity induce the biphasic tryptic inactivation pattern in the presence of K+. Their order of effectivity is inversely related to the rate constant of the second (slow) phase of inactivation. In the presence of K+ and either ITP or GTP tryptic inactivation remains monoexponential, indicating that these nucleotides cannot drive the E2K----E1K transition. Tryptic inactivation has been compared with tryptic fragmentation of the alpha-subunit (apparent mol. wt. 94 kDa) of (Na+ + K+)-ATPase. In the E1 conformation (Na+ present) a 71 kDa fragment is formed during the second phase of inactivation. In the E2 conformation (K+ present) the alpha-subunit is split to fragments of 41 and 52 kDa. In the presence of K+ and ATP, ADP, AdoPP[NH]P or CTP the 71 kDa fragment is formed in amounts which decrease in the order ATP approximately equal to ADP greater than AdoPP[NH]P greater than CTP. In the presence of K+ and AMP, ITP or GTP the 71 kDa fragment is absent and only the E2 fragments are formed. From these and literature data we arrive at a specificity order for the E2K----E1K transition of ATP greater than ADP greater than AdoPP[NH]P greater than CTP greater than ITP = GTP = AMP. The same order holds for K+ transport in the K+-K+ exchange and for overall hydrolytic activity (Na+ + K+ present) with the natural nucleoside triphosphates as substrates. This marks the E2K----E1K transition as the step in the reaction mechanism that determines nucleotide specificity for (Na+ + K+)-activated hydrolysis and coupled cation transport.

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme.

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.