PubMed Health⌕ Search

Biomedical subjects

A Enyedi

Publications and source records attributed to A Enyedi.

At least 19 recordsLinked to original sources
PubMed2001

The plasma membrane calcium pump displays memory of past calcium spikes. Differences between isoforms 2b and 4b.

To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k(act) increased with Ca(2+) along a sigmoidal curve. At all Ca(2+) concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca(2+) concentration and also tested these simulations experimentally; PMCA was activated by 500 nm Ca(2+) and then exposed to 50 nm Ca(2+) for 10 to 150 s, and then Ca(2+) was increased again to 500 nm. During the second exposure to 500 nm Ca(2+), the activity reached steady state faster than during the first exposure at 500 nm Ca(2+). This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nm Ca(2+). The peptide inhibited the activity of PMCA2b when the exposure to 50 nm Ca(2+) was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca(2+). The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca(2+) more quickly in subsequent spikes after an initial activating spike.

Calcium↗
PubMed2001

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Animals↗
PubMed2001

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells.

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.

Animals↗
PubMed2000

Plasma membrane Ca(2+) pump isoform 3f is weakly stimulated by calmodulin.

Isoform 3f of the plasma membrane Ca(2+) pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K(d) of 15 nM) than the corresponding peptide of isoform 2b (K(d) value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.

Amino Acid Sequence↗
PubMed1999

The rate of activation by calmodulin of isoform 4 of the plasma membrane Ca(2+) pump is slow and is changed by alternative splicing.

A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca(2+) pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 microM free Ca(2+)) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of full-length 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca(2+) spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca(2+) signals.

Alternative Splicing↗
PubMed1999

Protein kinase C phosphorylates plasma membrane Ca2+ pump isoform 4a at its calmodulin binding domain.

Phosphorylation by protein kinase C of isoform 4a of the human plasma membrane Ca2+ pump (hPMCA4a) was studied using the COS cell expression system. Phosphorylation of several truncated mutants of hPMCA4a indicated that a single phosphorylation site lies in a region between residues 1113 and 1125. This region is within the calmodulin binding domain and contains a single phosphorylatable residue, serine 1115. Converting this serine to an alanine diminished phosphorylation greatly. Phosphorylation, done in the absence of calmodulin, did not affect subsequent calmodulin binding, but previous binding of calmodulin did inhibit phosphorylation. Moreover, no significant shift in the calmodulin response curve of hPMCA4a was observed when phosphorylation was mimicked by converting serine 1115 to an acidic residue. The calmodulin binding domain of hPMCA4a is much longer than other calmodulin binding domains and has been suggested to consist of two binding lobes interrupted by a short nonbinding region. The findings of this study indicate that serine 1115 is the residue phosphorylated by protein kinase C, and that it lies within the nonbinding region of the calmodulin binding domain.

Amino Acid Sequence↗
PubMed1998

Modulation of the plasma membrane Ca2+ pump.

The plasma membrane calcium pump, which ejects Ca2+ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation. Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms 1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced by another isoform.

Amino Acid Sequence↗
PubMed1998

Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets.

Antibodies 5F10 and JA3 (raised against the erythrocyte Ca2+ pump) were used to identify hPMCA4b as the major form of the plasma membrane Ca2+ pump in human platelets and in three human megakaryoblastoid cell lines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T. Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316: 353-359]. In addition to hPMCA4b, hPMCA1b was also detected in the megakaryoblastoid cells by using isoform-specific polyclonal antibodies. The apparent size of this isoform, however, was smaller than that seen in HeLa and COS-7 cell membranes indicating the presence of a modified form of hPMCA1b. In platelets, no evidence of the expression of hPMCA1b could be found. The amount of PMCA in these cells was compared with that of the constitutive form of the sarco/endoplasmic reticulum Ca2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low level of the plasma membrane Ca2+ pump was found in platelets while in their precursor cells the expression of this Ca2+ pump was much more abundant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change substantially upon megakaryocytic differentiation.

Animals↗
PubMed1997

Protein kinase C phosphorylates the "a" forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin.

Phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca2+ pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed.

Amino Acid Sequence↗
PubMed1997

Plasma membrane Ca2+ pump in rat brain. Patterns of alternative splices seen by isoform-specific antibodies.

The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.

Alternative Splicing↗
PubMed1997

Plasma membrane Ca2+ pump isoforms 2a and 2b are unusually responsive to calmodulin and Ca2+.

The full-length a and b variants of the rat plasma membrane calcium pump, isoform 2 (rPMCA2a and rPMCA2b), were constructed and expressed in COS-7 cells. To characterize these isoforms, calcium transport was determined in a microsomal fraction. Both rPMCA2a and rPMCA2b had a much higher affinity for calmodulin than the corresponding forms of hPMCA4, and rPMCA2b had the highest affinity among the isoforms that have been tested so far. When analyzed at a relatively high calmodulin concentration, rPMCA2b and, to a lesser extent, rPMCA2a showed higher apparent calcium affinity; i.e. they were more active at lower Ca2+ concentrations than hPMCA4b. This indicates that these two variants of rat isoform 2 will tend to maintain a lower free cytosolic Ca2+ level in cells where they are expressed. Both variants also showed a higher level of basal activity (in the complete absence of calmodulin) than hPMCA4b, a property which would reinforce their ability to maintain a low free cytosolic Ca2+ concentration. Experiments designed to determine the source of the higher apparent Ca2+ affinity of rPMCA2b showed that it came from the properties of the carboxyl terminus, rather than from any difference in the catalytic core.

Animals↗
PubMed1996

Protein kinase C activates the plasma membrane Ca2+ pump isoform 4b by phosphorylation of an inhibitory region downstream of the calmodulin-binding domain.

The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.

Amino Acid Sequence↗
PubMed1996

Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies.

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

Alternative Splicing↗
PubMed1996

Plasma membrane calcium pump isoform 4a has a longer calmodulin-binding domain than 4b.

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.

Adenosine Triphosphate↗
PubMed1995

Mutants in the putative nucleotide-binding region of the plasma membrane Ca(2+)-pump. A reduction in activity due to slow dephosphorylation.

Mutants of individual residues of the plasma membrane Ca(2+)-pump were made in the highly conserved region that (in related P-type ATPases) has been associated with nucleotide binding. Alteration of the strictly conserved Asp672 to Glu nearly eliminated the ability of the pump to transport Ca2+, while alteration at Val674, Arg675, and Lys686 reduced the activity. High levels of ATP (25 mM) did not overcome the reduced activity, indicating that it could not be due to a reduction in the affinity for ATP. Effects not directly related to ATP binding seemed to result from mutations in this area. For instance, the amount of phosphorylated intermediate in the most severely inhibited mutant, Asp672-->Glu, was nearly as high as that in the wild type, a much larger amount of phosphorylated intermediate than was expected from its low activity. However, the rate of decomposition of this intermediate was much slower than that of the wild type, indicating that the inhibition of this mutant resulted from an inhibition of the E approximately P-->E step in the enzyme cycle.

Adenosine Triphosphatases↗
PubMed1995

Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase.

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882.

Amino Acid Sequence↗
PubMed1994

Controlled proteolysis of Ca(2+)-ATPases in human platelet and non-muscle cell membrane vesicles. Evidence for a multi-sarco/endoplasmic reticulum Ca(2+)-ATPase system.

Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.

Blood Platelets↗