PubMed Health⌕ Search

Biomedical subjects

A R Penheiter

Publications and source records attributed to A R Penheiter.

6 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

Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2.

The deafwaddler mutant in mice was the first spontaneous mutant discovered in the plasma membrane Ca(2+) pump (PMCA) [Street, V.A. et al., 1998, Nat. Genet. 19, 390-394]. A nucleotide substitution in deafwaddler results in a Gly to Ser transition at amino acid 283 in the small cytoplasmic loop of PMCA isoform 2 (PMCA2). PMCA2 is abundant in the stereocilia of auditory and vestibular hair cells, neurons of the spiral ganglion, and participates in inner ear development. Mice that are homozygous for deafwaddler are deaf and have poor balance. However, the balance and hearing disorders of the deafwaddler mice appear to be less severe than homozygotes for a functionally null frameshift mutant or homozygous PMCA2 knockout mice, suggesting that deafwaddler PMCA2 retains some biological activity. To examine the enzymic effects of the deafwaddler mutant, PMCA2 wild-type and deafwaddler were produced by transient expression in COS cells as well as baculovirus-mediated expression in Sf9 insect cells. Membrane preparations were assayed for calcium transport and ATPase activity. No significant differences in the regulation by calmodulin of the wild-type and deafwaddler PMCA2b were found. Steady-state transport assays and pre-steady-state ATPase assays of these two proteins revealed that the K(0.5) for Ca(2+), K(0.5) for calmodulin, degree of activation by calmodulin and rate of activation by Ca-calmodulin were nearly identical. However, calcium transport of the deafwaddler pump was reduced to 30% of the wild-type activity. Although calcium transport activity was reduced in the deafwaddler pump, total phosphoenzyme formation from ATP was slightly higher for deafwaddler than for wild-type. 50 microM LaCl3 (which blocks the E(1)P to E(2)P conformational transition) increased the steady-state level of phosphoenzyme 3-fold for the wild-type but had no effect on the deafwaddler. Taken together, the kinetic data suggest that the deafwaddler mutation affects PMCA2 by slowing the E(1)P to E(2)P transition, resulting in approximately 70% reduction in the PMCA2-mediated Ca(2+) export.

Adenosine Triphosphate↗
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↗
PubMed1998

Purification and characterization of a soybean root nodule phosphatase expressed in Pichia pastoris.

Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5'-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In order to perform directed mutagenesis and more extensive biochemical characterization, a means of producing recombinant ACP was needed. Several attempts were made to express ACP in Escherichia coli, but all conditions employed resulted in protein that was found entirely in inclusion bodies, and resolubilization experiments were unsuccessful. Therefore, the methyltrophic yeast Pichia pastoris was chosen as a eukaryotic expression host. The coding sequence of ACP was cloned into the pPIC9 vector to create a fusion with the yeast alpha mating factor secretion signal. The ACP:pPIC9 construct was integrated into P. pastoris strain GS115. Expression of ACP was under the control of an alcohol oxidase methanol-inducible promoter. Methanol induction resulted in secretion of ACP to a level of 10 mg/L. The recombinant ACP was purified 550-fold to homogeneity by phenyl-Sepharose, hydroxyapatite, and MonoS chromatography. The purified enzyme had Km values of 0.08 and 0.12 for 5'-AMP and 5'-GMP. These values were similar to those obtained for the native ACP heterodimer purified from soybean (0.08 and 0.15 mM for 5'-AMP and 5'-GMP). The specific activity of the recombinant enzyme for all substrates tested was 1.6- to 1.8-fold higher than the values for the purified soybean heterodimer.

Acid Phosphatase↗
PubMed1997

Soybean root nodule acid phosphatase.

Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.

Acid Phosphatase↗