Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.
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PMID:A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080. 1006 37

Kidneys of full-term newborn humans and animals conserve potassium (K+), a condition essential for growth. The cortical collecting duct (CCD) is uniquely adapted to accomplish this task early in life. CCDs isolated from newborn rabbits and microperfused in vitro show no net K+ secretion until after the third week of life; in contrast, segments isolated from adult animals secrete net K+ at high rates. The magnitude and direction of net K+ transport in the CCD reflect the balance of opposing fluxes of K+ secretion and K+ absorption mediated by principal and intercalated cells, respectively. The absence of net K+ secretion in the CCD early in life may thus be caused by a limited capacity of principal cells for K+ secretion and/or an excess of K+ absorption by intercalated cells. Recent studies provide data to support both possibilities. Patch-clamp analysis detects few conducting apical K+-secretory channels in neonatal principal cells, whereas fluorescent functional assays identify significant activity of the apical hydrogen, potassium adenosine triphosphatase (H+,K+-ATPase), a pump that reabsorbs K+ in exchange for H+s, in adjacent intercalated cells. Under conditions prevailing in vivo, the sum of the fluxes mediated by these two cell types likely contributes to the relative K+ retention characteristic of the neonatal kidney.
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PMID:Regulation of potassium transport in the maturing kidney. 1019 48

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.
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PMID:The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes. 1020 69

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.
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PMID:The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon. 1039 9

Omeprazole and pantoprazole are known to be irreversible, SH-acting inhibitors of gastric H+,K+-adenosine triphosphatase (H+,K+-ATPase). Both drugs concentration-dependently and pH-dependently inhibited K+-dependent p-nitrophenyl phosphatase (K+-pNPPase) activity in purified rabbit gastric microsomes. The potency of omeprazole was about three times that of pantoprazole in the pH ranges tested. Both drugs also inhibited acid secretion, as determined by [14C]aminopyrine accumulation in isolated rabbit gastric glands, with the potency ratio being about 5 (omeprazole over that of pantoprazole). Under conditions in which acid secretion was inhibited completely by the drugs, the total K+-pNPPase activity in the digitonin-permeabilized glands was scarcely reduced, showing an apparent discrepancy between the acid secretion and the proton pump activity. The isolated glands were stimulated with secretagogues for 30 min in the presence of the inhibitors, homogenized, and then separated into fractions in which K+-pNPPase activity was measured. Omeprazole exclusively inhibited the activity in the low-speed fraction, which was rich in the apical membranes, whereas pantoprazole did not inhibit activity in any fraction. When the time of treatment with the inhibitors was increased up to 5 hr, the inhibition of the total K+-pNPPase activity in the glands reached a plateau at an inhibition rate lower than 50% within 2 hr. This suggested that no continuous recycling of the proton pump was occurring during stimulation. The inhibitory effect of both drugs on the permeabilized gland preparation was less potent than that on the purified enzyme, especially at the higher pH, and it appeared to be partially reversible. The extent of the reduction in potency was more prominent for pantoprazole. It is concluded that a lower amount of proton pump activity needs to be inhibited by pantoprazole than by omeprazole to achieve the same extent of acid secretion inhibition. This appears to be due to the nature of pantoprazole, i.e. the requirement of low pH for activation and the partial reversibility of the inhibition.
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PMID:Correlation between acid secretion and proton pump activity during inhibition by the proton pump inhibitors omeprazole and pantoprazole. 1048 39

The H+,K+-ATPases belong to the X+,K+-ATPase subfamily of P-type cation-transporting ATPases. While these H+,K+-ATPase isoforms share approximately 60%-70% amino acid identity, they exhibit discrete kinetic and pharmacological properties. The colonic alpha isoform (HKalpha2) is insensitive to Sch-28080, an inhibitor of the gastric H+,K+-ATPase, and is sensitive to high concentrations of ouabain. This profile contrasts with the sensitivities attributed to HKalpha2 in transport studies. HKalpha2 mRNA and protein abundance appear to be both site-specifically upregulated in response to chronic hypokalemia, and have been localized to the outer and inner medulla. To reconcile expressed sensitivities with those reported in vitro in isolated tubules and cells in culture, it requires transformation of the expressed insensitivity of the colonic H+,K+-ATPase to Sch-28080. Although a "unique" beta subunit has been reported recently, this beta subunit ("betac"), is identical at the amino acid level to the recently cloned beta3-Na+,K+-ATPase. Moreover, while HKalpha2 can assemble indiscriminately with any X+,K+-ATPase beta subunit, HKalpha2 has been reported to assemble stably with beta1-Na+,K+-ATPase in the renal medulla and in the distal colon. It is conceivable that subunit assembly could be tissue-specific and might respond to different physiological and pathophysiological stimuli. Recent studies have suggested that the H+,K+-ATPase is both Na+-dependent and localized to the apical membrane in the distal colon. Future studies will be needed to resolve these discrepancies by determining if a unique, yet undiscovered H+,K+-ATPase isoform exists in the kidney, or if posttranslational modifications of the alpha and/or beta-subunits could account for these functional diversities.
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PMID:Contrasting functional and regulatory profiles of the renal H+,K+-ATPases. 1051 79

The H+,K+-ATPases comprise a group of integral membrane proteins that belong to the X+,K+-ATPase subfamily of P-type cation-transporting ATPases. Although these H+,K+-ATPase isoforms share approximately 60-70% amino acid identity, they exhibit discrete kinetic and pharmacological properties when expressed in heterologous systems. HK alpha2 has been categorized by its insensitivity to Sch-28080, an inhibitor of the gastric H+,K+-ATPase, and partial sensitivity to ouabain, an inhibitor of the Na+,K+-ATPase. This functional profile contrasts with the pharmacological sensitivities ascribed to HK alpha2 in transport studies in rat isolated medullary collecting ducts perfused in vitro and in mouse medullary collecting duct cell lines. HK alpha2 mRNA and protein abundance appears to be both tissue and site-specifically upregulated in response to chronic hypokalemia. This regulatory response has been localized to the outer and inner medulla. To reconcile these expressed sensitivities to those reported in vitro in isolated tubules and cells in culture, it would be necessary to invoke modification of the pharmacologic insensitivity of the colonic H+,K+-ATPase to Sch-28080. Although a 'unique' beta-subunit has been reported recently, this beta-subunit (beta(c)) is identical at the amino acid level to the recently cloned beta3-Na+,K+-ATPase. Moreover, while HK alpha2 can assemble indiscriminately with any X+,K+-ATPase beta-subunit, HK alpha2 has been reported to assemble stably with beta1-Na+,K+-ATPase in the renal medulla and in the distal colon. It remains conceivable that subunit assembly could be tissue specific and might respond to different physiological and pathophysiological stimuli. Furthermore, recent studies have suggested that the H+,K+-ATPase is both Na+-dependent and localized to the apical membrane in the distal colon. Therefore, future studies will need to resolve these discrepancies by determining if a unique, yet undiscovered H+,K+-ATPase isoform exists in kidney, or if post-translational modifications of the alpha- and/or beta-subunits could account for these functional diversities.
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PMID:H+,K+-ATPase. 1054 Dec 23

To study the role of Glu795offresent in the fifth transmembrane domain of the alpha-subunit of gastric H+,K+-ATPase, several mutants were generated and expressed in Sf9 insect cells. The E795Q mutant had rather similar properties as the wild-type enzyme. The apparent affinity for K+ in both the ATPase reaction and the dephosphorylation of the phosphorylated intermediate was even slightly enhanced. This indicates that the carbonyl group of Glu795 is sufficient for enzymatic activity. This carbonyl group, however, has to be at a particular position with respect to the other liganding groups, since the E795D and E795N mutants showed a strongly reduced ATPase activity, a lowered apparent K+ affinity, and a decreased steady-state phosphorylation level. In the absence of a carbonyl residue at position 795, the K+ sensitivity was either strongly decreased (E795A) or completely absent (E795L). The mutant E795L, however, showed a SCH 28080 sensitive ATPase activity in the absence of K+, as well as an enhanced spontaneous dephosphorylation rate, that could not be further enhanced by K+, suggesting that this mutant mimicks the filled K+ binding pocket. The results indicate that the Glu795 residue is involved in K+-stimulated ATPase activity and K+-induced dephosphorylation of the phosphorylated intermediate. Glu795 might also be involved in H+ binding during the phosphorylation step, since the mutants E795N, E795D, and E795A showed a decrease in the phosphorylation rate as well as in the apparent ATP affinity in the phosphorylation reaction. This indicates that Glu795 is not only involved in K+ but might also play a role in H+ binding.
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PMID:The carbonyl group of glutamic acid-795 is essential for gastric H+,K+-ATPase activity. 1068 13

The effects of site-directed mutagenesis were used to explore the role of residues in M4 on the apparent Ki of a selective, K+-competitive inhibitor of the gastric H+,K+ ATPase, SCH28080. A double transfection expression system is described, utilizing HEK293 cells and separate plasmids encoding the alpha and beta subunits of the H+,K+-ATPase. The wild-type enzyme gave specific activity (micromoles of Pi per hour per milligram of expressed H+,K+-ATPase protein), apparent Km for ammonium (a K+ surrogate), and apparent Ki for SCH28080 equal to the H+, K+-ATPase purified from hog gastric mucosa. Amino acids in the M4 transmembrane segment of the alpha subunit were selected from, and substituted with, the nonconserved residues in M4 of the Na+, K+-ATPase, which is insensitive to SCH28080. Most of the mutations produced competent enzyme with similar Km,app values for NH4+ and Ki,app for SCH28080. SCH28080 affinity was decreased 2-fold in M330V and 9-fold in both M334I and V337I without significant effect on Km,app. Hence methionine 334 and valine 337 participate in binding but are not part of the NH4+ site. Methionine 330 may be at the periphery of the inhibitor site, which must have minimum dimensions of approximately 16 x 8 x 5 A and be accessible from the lumen in the E2-P conformation. Multiple sequence alignments place the membrane surface near arginine 328, suggesting that the side chains of methionine 334 and valine 337, on one side of the M4 helix, project into a binding cavity within the membrane domain.
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PMID:Effects of mutations in M4 of the gastric H+,K+-ATPase on inhibition kinetics of SCH28080. 1071 20

Hinesol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), strongly inhibited H+,K+-ATPase activity with a IC50 value of 5.8x10(-5) M. It also inhibited Na+,K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, and H+-ATPase activities, although the inhibition rate was lower. No effects on alkaline or acid phosphatase activities were observed. The mechanism by which hinesol inhibited H+,K+-ATPase activity was studied in detail. The inhibition was uncompetitive with respect to ATP, and it increased as the Mg2+ concentration was raised, whereas it was not affected by the K+ concentration. The activity of K+-dependent p-nitrophenyl phosphatase (K+-pNPPase), a partial reaction of H+,K+-ATPase, was inhibited by hinesol noncompetitively with respect to pNPP (IC50 value of 1.6x10(-4) M), and competitively with respect to K+, whereas it was not affected by the Mg2+ concentration. These results suggest that hinesol is a relatively specific inhibitor of H+,K+-ATPase. It appears that hinesol reacts with enzyme in the E1 state in the presence of ATP and Mg2+ and forms the complex hinesol-H+ E1-ATP or hinesol x E1-P, blocking the conformational change to the E2 state. Furthermore, hinesol enhanced the inhibitory effect of omeprazole on H+,K+-ATPase, and the inhibitory site of hinesol was different from that of omeprazole. The effect of So-jutsu as an anti-gastric ulcer agent may be ascribed to the inhibitory effect of hinesol on H+,K+-ATPase activity.
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PMID:Inhibition of H+,K+ -ATPase by hinesol, a major component of So-jutsu, by interaction with enzyme in the E1 state. 1071 47


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