Acylphosphatase induced modifications in the functional properties of erythrocyte membrane sodium pump

doi:10.1016/0005-2736(93)90311-M

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 1147, Issue 1, 8 April 1993, Pages 19-26

https://doi.org/10.1016/0005-2736(93)90311-M Get rights and content

Abstract

Human red cell acylphosphatase actively hydrolyzes the Na⁺/K⁺-ATPase phosphoenzyme from erthrocyte membrane. This effect occurred with amounts of acylphosphatase (up to 10 units/mg membrane protein) within the physiological range, and the low value of the apparent K_m (0.147±0.050μM) indicates that the enzyme has a high affinity for this substrate. When added at the above concentration to inside out vesicles from human erythrocytes, acylphosphatase significantly enhanced the rate of strophantidine-sensitive ATP hydrolysis. The same amounts of acylphosphatase stimulated, although to a lower extent, the rate of ATP-dependent ²²Na⁺ influx (normal efflux). Thus, the calculated stoichiometry for Na⁺/ATP was 2.68 in the absence of acylphosphatase and 1.06 in the presence of 10 units/mg vesicle protein of the enzyme. Conversely, acylphosphatase addition strongly decreased the rate of ATP-dependent ⁸⁶Rb⁺ (K⁺) efflux (normal influx) which, with 10 units/mg vesicle protein, was almost suppressed. As a consequence, the Na⁺/Rb⁺ ratio, calculated as 1.52 in the absence of acylphosphatase rose to 72.5 in the presence of 10 units/mg vesicle protein of this enzyme. These results suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase ‘uncouples’ erythrocyte membrane Na⁺, K⁺ pump. Possible mechanisms for this effect are discussed.

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Cited by (22)

High resolution structure of Vibrio cholerae acylphosphatase (VcAcP) cage: Identification of drugs, location of its binding site and engineering to facilitate cage formation
2020, Biochemical and Biophysical Research Communications
Citation Excerpt :
Acylphosphatase (AcP; EC 3.6.1.7) is a widespread small (10 kDa) cytosolic enzyme consists of two helices and five antiparallel β-strands. It catalyses the hydrolysis of carboxyl-phosphate bonds of acylphosphates [5] and reported to form oligomer(s) in solution [6,7]. We have previously reported that Vibrio cholerae O395 acylphosphatase (VcAcP) readily form a helix mediated trimer (HT) in solution [8].
Protein cages have recently emerged as an extraordinary drug-delivery system due to its biocompatibility, biodegradability, low toxicity, ease to manipulate and engineer. We have reported earlier the formation and architecture of a do-decameric cage-like architecture of Vibrio cholerae acylphosphatase (VcAcP) at 3.1 Å. High resolution (2.4 Å) crystal structure of VcAcP cage, reported here, illuminates a potential binding site for sulphate/phosphate containing drugs whereas analysis of its subunit association and interfaces indicates high potential for cage engineering. Tryptophan quenching studies indeed discloses noteworthy binding with various sulphate/phosphate containing nucleotide-based drugs and vitamin B₆ (PLP) demonstrating that exterior surface of VcAcP protein cage can be exploited as multifunctional carrier. Moreover, a quadruple mutant L30C/T68C/N40C/L81C-VcAcP (QM-VcAcP) capable to form an intricate disulphide bonded VcAcP cage has been designed. SEC, SDS-PAGE analysis and DLS experiment confirmed cysteine mediated engineered VcAcP cage formation.
Investigation of Na<sup>+</sup>,K<sup>+</sup>-ATPase on a solid supported membrane: The role of acylphosphatase on the ion transport mechanism
2003, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
More recently, Nassi and coworkers reported that AcP can act not only on the abovementioned low molecular weight substrates, but also on the acylphosphorylated intermediates of various transport ATPases, including Na+,K+-ATPase [6,7]. Although this effect was observed with a denatured phosphoenzyme [6], the same authors demonstrated the occurrence of a molecular interaction between AcP and native, undenatured Na+,K+-ATPase by dot immunobinding analysis [7]; they also found that AcP addition modifies the functional properties of this active transport system by increasing the rate of ATP hydrolysis and the Na+/K+ exchange ratio with respect to the well-documented 3/2 value. Based on these results, AcP has been proposed as a potential modulator of the sodium pump [7].
Charge translocation by Na⁺,K⁺-ATPase was investigated by adsorbing membrane fragments containing Na⁺,K⁺-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na⁺,K⁺-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na⁺ and K⁺ ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme α-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na⁺,K⁺-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na⁺ but not of K⁺, the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na⁺,K⁺-ATPase.
Identification and characterization of genes differentially expressed in the testis/vas deferens of the fed male tick, Amblyomma hebraeum
2002, Insect Biochemistry and Molecular Biology
Most ixodid ticks must feed for at least a few days to complete gonad maturation. Substances produced by the mature male gonad, and carried in the spermatophore, induce physiological changes in the female that lead to engorgement and oviposition. To begin defining the molecular phenotype at this stage of male development, we differentially cross-screened a cDNA library made from the testis and vas deferens of fed ticks (Amblyomma hebraeum Koch) and isolated 35 genes that were putatively up-regulated in tissues of fed compared to unfed animals. While the majority of these were novel, two clones, AhT/VD16 and AhT/VD146, yielded homologies (53 and 44%) to known genes (acylphosphatse and 9.0 kD Drosophila melanogaster basic protein, respectively). Results of Northern blot analysis of AhT/VD16 and AhT/VD146 demonstrate that both clones hybridized with mRNA transcripts that were up-regulated in the testis/vas deferens of fed compared to unfed males. In addition, hybridization of clone AhT/VD16 to water strider (Gerris argentatus) genomic DNA, and sequence similarities to mammalian acylphosphatase, suggest that it represents an evolutionarily conserved sequence.
Acylphosphatase is a strong apoptosis inducer in HeLa cell line
2000, Molecular Cell Biology Research Communications
Acylphosphatase (AcP) is a low-molecular-weight protein widely distributed in many vertebrate tissues with a yet unknown physiologic function. To study the in vivo behavior of AcP, HeLa cells were transiently transfected with a vector expressing the AcP/EGFP fusion protein. Analysis of the transfected cells showed a high level of cellular death in cells expressing the AcP/EGFP fusion protein with respect to control cells expressing EGFP alone. Flow cytometry and time lapse analysis of AcP/EGFP transfected cells evidenced a typical pattern of apoptosis. Surprisingly, cells transfected with a mutated form of AcP, with negligible in vitro acylphosphatase activity, undergo apoptosis as well as cells transfected with wild-type protein, suggesting that the physiologic role of AcP could be not related to this catalytic activity.
Drosophila melanogaster acylphosphatase : A common ancestor for acylphosphatase isoenzymes of vertebrate species
1998, FEBS Letters
An open reading frame encoding a putative acylphosphatase was found in Drosophila melanogaster. The corresponding gene product shows 40% identity and 22 additional amino acid residues at the C-terminus as compared to muscle- and common-type human acylphosphatases. Moreover, all the residues involved in the catalytic mechanism of vertebrate enzymes are conserved in the D. melanogaster acylphosphatase. The D. melanogaster protein and a deletion mutant, similar in length to vertebrate acylphosphatases, were produced by cloning the corresponding cDNA in Escherichia coli. The wild-type enzyme is a protein with a well-established three-dimensional fold and a markedly reduced conformational stability as compared to vertebrate isoenzymes. The specific activity of the enzyme is significantly lower than that found in vertebrate enzymes though the substrate binding capability is basically unaltered. The deletion of 22 residues does not cause a significant change in k_cat, while affecting the apparent binding parameters. This work suggests that the genes encoding the vertebrate enzymes originate from an ancestor gene by duplication and subsequent evolution.
Crystal structure of common type acylphosphatase from bovine testis
1997, Structure
Background: Acylphosphatase (ACP) is a low molecular weight phosphomonohydrolase catalyzing with high specificity the hydrolysis of the carboxyl-phosphate bond present in acylphosphates. The enzyme is thought to regulate metabolic processes in which acylphosphates are involved, such as glycolysis and the production of ribonucleotides. Furthermore the enzyme is capable of hydrolyzing the phospho-aspartyl intermediate formed during the action of membrane pumps such as (Ca²⁺+Mg²⁺) ATPase. Although the tertiary structure of a muscle ACP has been determined by NMR spectroscopy, little is known about the catalytic mechanism of ACP and further structures might provide an increased understanding.
Results: The structure of ‘common type’ ACP from bovine testis has been determined by X-ray crystallography to a resolution of 1.8 Å. The structure has been refined to an R factor of 17.0 % using all data between 15 and 1.8 Å. The binding of a sulphate and a chloride ion in the active centre allows a detailed description of this site. The overall protein folds of common type and muscle ACP are similar but their loops have very different conformations. These differences, in part, are probably caused by the binding of the ions in the active site of the common type form. The phosphate-binding loop of ACP shows some remarkable similarities to that of low molecular weight protein tyrosine phosphatase.
Conclusions: The active site of ACP has been located, enabling a reaction mechanism to be suggested in which the phosphate moiety bound to Arg23 acts as a base, abstracting a proton from a nucleophilic water molecule liganded to Asn41. The transition-state intermediate is stabilized by the phosphate-binding loop. We suggest the catalysis to be substrate assisted, which probably explains why this enzyme can only hydrolyze acylphosphates.

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Regular paperAcylphosphatase induced modifications in the functional properties of erythrocyte membrane sodium pump

Abstract

Methods Enzymol.

FEBS Lett.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Mech. Ageing Dev.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Experentia (Basel)

G. Biochim.

Ital. J. Biochem.

Regular paper
Acylphosphatase induced modifications in the functional properties of erythrocyte membrane sodium pump