The molecular basis of the modulation of the plasma membrane calcium pump by calmodulin.
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Amino Acid Sequence↗
Biomedical subjects
J Krebs
Publications and source records attributed to J Krebs.
Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.
Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).
Affinity Labels↗
Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca2+ pump.
The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides.
Amino Acid Sequence↗
Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump.
Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with the calmodulin binding domain, supporting the suggestion (Benaim et al., 1984) that the latter, or a portion of it, may act as a natural inhibitor of the pump.
Animals↗
Structural details of the interaction of calmodulin with the plasma membrane Ca2(+)-ATPase.
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Amino Acid Sequence↗
Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain.
The activity of the membrane-bound and the purified erythrocyte Ca2+-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatures and in the purified ATPase digested at higher calpain/ATPase ratios, the ATPase became fully activated. The membrane-bound and the purified 138-kDa ATPase were converted by calpain to a fragment of approximately 124 kDa which still bound CaM and could be isolated on CaM columns when proteolysis occurred slowly but not when it occurred rapidly. Carboxypeptidase digestion of the purified enzyme and of its fragment of about 124 kDa has shown that calpain attacked the CaM-binding domain near the C terminus of the ATPase. This has also been supported by digestion of the purified enzyme and of its fragment of about 124 kDa. A first cut occurred in the middle of the domain producing a fragment of about 14 kDa and a (CaM-binding) fragment of about 124 kDa. A second cut closer to the N terminus of the domain also produced a fragment of about 124 kDa and accounted for the loss of CaM binding at prolonged times of incubation of the ATPase with calpain.
Amino Acid Sequence↗
Multiple divergent mRNAs code for a single human calmodulin.
The isolation of a novel complementary DNA (cDNA) clone coding for human calmodulin (CaM) is reported. Although it encodes a protein indistinguishable from the only known higher vertebrate calmodulin, its nucleotide sequence varies extensively from that of two previously reported human CaM cDNAs (Wawrzynczak and Perham, 1984; SenGupta et al., 1987). Only 82 and 81% identity, respectively, is found between the newly isolated and the two known human mRNAs in their coding regions. No striking homology is present in their noncoding regions. Codon usage in the three CaM mRNAs is also surprisingly divergent. A 2.3-kilobase mRNA corresponding to the newly isolated clone is expressed to varying extents in several human tissues, together with an approximately 0.8-kilobase mRNA species presumably arising from alternative polyadenylation of the same primary transcript. The results indicate that the human genome contains at least three divergent CaM genes that are under selective pressure to encode an identical protein while maintaining maximally divergent nucleotide sequences. Partial characterization of a genomic clone specifying the 3' portion of the newly identified CaM mRNA shows that this gene contains introns at identical positions as the previously characterized bona fide vertebrate CaM genes. Evolutionary implications of the presence of a CaM multigene family are discussed.
Amino Acid Sequence↗
Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes.
Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.
Amino Acid Sequence↗
Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both HIV type 1 and type 2 proviral sequences by using polymerase chain reaction.
Sera from persons seroreactive to both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), by whole-virus (VEIA) enzyme immunoassays (EIAs) for each virus, were selected from a seroprevalence study of 944 persons in Abidjan, Cote d'Ivoire, West Africa, in 1987. These sera were subsequently tested for HIV-1 and HIV-2 antibody specificity by type-specific peptide EIAs (PEIA) and western blot (WB) analysis for both viruses. Peripheral blood monocytes (PBMCs) from representative individuals were cultured in the presence of phytohemagglutinin-stimulated normal donor PBMCs. These cultures were periodically monitored for HIV-1 and HIV-2 proviral sequences by using the selective DNA amplification technique polymerase chain reaction (PCR). As an outgrowth of this study, we report the case of a person dually reactive by various serological techniques in whom proviral sequences from HIV-1 and HIV-2 were detected by PCR. This is the first confirmed case of a mixed HIV-1 and HIV-2 infection in a single individual.
Acquired Immunodeficiency Syndrome↗
Bed rest effect on extremity muscle torque in healthy men.
The purpose of this study was to assess the effect of bed rest on appendicular muscle torque. Fifteen healthy male volunteers between the ages of 21 and 54 participated in the study. The subjects were put on five weeks of strict horizontal bed rest. They were allowed to sit up for bowel movements only; however, no lower extremity weight-bearing was allowed. During bed rest, the subjects were required to ingest a fixed nutritionally adequate diet. Data for muscle torque were obtained using a Cybex II dynamometer, Upper Body Exercise Table (UBXT), and a data analysis computer (HUMAC). The subjects were tested on Cybex at the speed of 60 degrees/sec during the week before starting bed rest and the day after starting reambulation. The muscle groups tested were soleus, gastrocnemius-soleus, dorsiflexors, knee flexors, knee extensors, elbow flexors, and elbow extensors. The torque in all groups but elbow extensors was reduced by 24%, 26%, 8%, 8%, 19%, and 7%, respectively. Student's t-test for paired means demonstrated significant loss of torque in all muscle groups except elbow extensors (p less than 0.05). The results of this study suggest that a strengthening exercise program should be administered to these patients while they are on bed rest, and that the earliest possible attempt should be made to ambulate patients after prolonged bed rest to minimize the loss of muscle torque.
Adult↗
Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin.
Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.
3',5'-Cyclic-AMP Phosphodiesterases↗
Conformational differences between the E1 and E2 states of the calcium adenosinetriphosphatase of the erythrocyte plasma membrane as revealed by circular dichroism and fluorescence spectroscopy.
Different conformational states of the purified plasma membrane Ca2+-ATPase from pig erythrocytes have been detected by circular dichroism (CD) and fluorescence spectroscopy. The helical content of the enzyme decreased by about 10% in the transition from the Ca2+ high-affinity form (10 microM free Ca2+ = E1 state) to the VO4(3-)-inhibited state (20 microM VO4(3-) = E2 state). The changes in the CD spectra did not show full reversibility upon reversing the E1-E2 transition, whereas those in the fluorescence spectra did. A temperature-dependent loss of alpha-helical content in the presence of Ca2+ was also observed. Intrinsic fluorescence measurements revealed an increase in fluorescence intensity upon addition of Ca2+. The change was fully reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The increase in fluorescence intensity was partly reversed by adding ATP, an effect which is suggested to correspond to the "Ca2+-occluded" form of the ATPase. The steady-state level of the fluorescence intensity was stable for several minutes in the presence of 100 microM ATP. By contrast, the decrease of fluorescence intensity induced by limiting concentrations of ATP (= 1 microM) was only transient, indicating the decomposition of the phosphorylated intermediate of the ATPase and the reestablishment of the Ca2+ high-affinity form of the enzyme.
Animals↗
Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia.
Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.
Animals↗
Spinal bone mineral after 5 weeks of bed rest.
Patients put at bedrest for medical reasons lose 1-2% of spinal bone mineral per week. Losses of this magnitude during even short-term space flights of a few months would pose a serious limitation and require countermeasures. The spinal bone mineral (L2-L4) was determined in 6 healthy males (precision = 2%) before and after 5 weeks of complete bed rest. Only one individual had a significant loss (3%) and the -0.9% mean change for the 6, was not significant (P = 0.06). The average negative calcium balance during the 5 weeks was 4 g or 0.36% of total body calcium, similar to that reported in other bed-rest studies. Spinal bone loss, however, in healthy bed-rested males is significantly less than reported for bed-rested patients, suggesting that a large loss of spinal bone mineral does not occur during space flight missions lasting 5 weeks or less.
Adult↗
Calmodulin-drug interaction. A fluorescence and flow dialysis study.
Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy and flow dialysis methods the stoichiometry of the binding of some of the drugs to calmodulin has been investigated. The number of Ca2+-dependent high affinity binding sites has been studied on trypsin fragments of calmodulin. Compound 12-114 was bound with high affinity in a Ca2+-dependent way to both halves of calmodulin, compound 200-737 recognized one high affinity binding site only in the C-terminal half of the molecule, whereas compound 36-079 demanded the intact protein to be able to interact with high affinity in a Ca2+-dependent manner.
Calcium-Transporting ATPases↗
Study of a hydrophobic site on bovine alpha-lactalbumin by labeling with [125I]-TID.
The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) labels apo-bovine alpha-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml-1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease the labeling of apo-alpha-lactalbumin with [125I]TID.
Animals↗
Clocks, pineals and compasses.
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Animals↗
Hormones and the pathophysiology of fibrocystic mastopathy: elevated luteinizing hormone levels.
Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, testosterone, estrone, and estradiol were measured weekly for 1 month in 12 menstruating women with fibrocystic mastopathy. LH levels were abnormally elevated. The remainder of hormone levels were essentially normal. Elevated serum LH may be secondary to hypothalamic-pituitary, ovulatory, or corpus luteum dysfunction. The roles of hormonal abnormalities in the pathogenesis of fibroycstic mastopathy are not known.
Adult↗