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R A Reithmeier

Publications and source records attributed to R A Reithmeier.

At least 73 records · Page 4Linked to original sources
PubMed1987

Amino acid sequence of rabbit fast-twitch skeletal muscle calsequestrin deduced from cDNA and peptide sequencing.

Partial amino acid sequence analysis of rabbit fast-twitch skeletal muscle calsequestrin permitted the construction of synthetic oligonucleotides that were used as both primers and probes for the synthesis and isolation of cDNAs encoding calsequestrin from neonatal rabbit skeletal muscle libraries. The cDNA sequence encodes a processed protein of 367 residues with a Mr of 42,435 and a 28-residue amino-terminal signal sequence. The deduced amino acid sequence agreed closely with the portions of the mature protein that were sequenced using standard protein sequencing. The neonatal protein, however, contains an acidic carboxyl-terminal extension not present in the adult protein, suggesting that the cDNA sequence may have arisen from an alternatively spliced neonatal transcript. A single transcript of 1.9-2.0 kilobases was seen in neonatal skeletal muscle mRNA. A glycosylation site and two potential phosphorylation sites were detected. Although the protein contains about two acidic residues for each Ca2+ bound, there is no repeating distribution of acidic residues and no evidence of EF hand structures. Hydropathy plots show no transmembrane sequences, and structural analyses suggest that less than half of the protein is likely to be highly structured. This sequence defines the characteristics of a class of high-capacity, moderate-affinity, Ca2+ binding proteins.

Amino Acid Sequence↗
PubMed1986

Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes.

Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37 degrees C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N-ethylmaleimide and p-chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or beta-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37 degrees C, pH 8.3 in sucrose-citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside.

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

31P and 35Cl nuclear magnetic resonance measurements of anion transport in human erythrocytes.

The exchange of anions across the erythrocyte membrane has been studied using 31P nuclear magnetic resonance (NMR) to monitor inorganic phosphate influx and 35Cl NMR to monitor chloride ion efflux. The 31P NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular pH to 6.4, while the intracellular pH was 7.3. The 35Cl NMR resonance for intracellular Cl- was so broad as to be virtually undetectable (line width greater than 200 Hz), while that of extracellular Cl-is relatively narrow (line width of about 30 Hz). The transports of Pi and Cl-were both totally inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a potent inhibitor of the band 3 protein. Since the 31P resonance of Pi varies with pH, intra- and extracellular pH changes could also be determined during anion transport. The extracellular pH rose and intracellular pH fell during anion transport, consistent with the protonated monoanionic H2PO4-form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPO24-form. The rates of Cl-efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The Cl-efflux was found to coincide with the influx of the monoanionic H2PO4-form of Pi.

Adult↗
PubMed1985

Conformation and stability of the anion transport protein of human erythrocyte membranes.

The conformation and stability of purified preparations of band 3, the anion transport protein of human erythrocyte membranes, and its constituent proteolytic subfragments have been studied by circular dichroism. Band 3, purified in the presence of the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8), had an alpha-helical content of 46%. Denaturation of purified band 3 with guanidine hydrochloride occurred in two phases, one reflecting much more resistance to denaturation than the other. Band 3 can be separated into two domains by limited in situ proteolytic cleavage. The carboxyl-terminal membrane-associated domain (Mr 55 000) purified in C12E8 contained 58% alpha-helix and was very resistant to denaturation by guanidine hydrochloride. The purified amino-terminal, cytoplasmic domain (Mr 41 000) contained 27% alpha-helix and was completely converted to a random-coil conformation by 3 M guanidine hydrochloride. The two phases of denaturation observed for intact band 3 corresponded to the two domains of the protein. Irreversible heat denaturation of purified band 3 occurred with half-maximal change in theta 222.5 at 48 degrees C. Covalent attachment of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate to band 3 had little effect on the circular dichroism spectra of band 3 or the membrane-associated domain but resulted in stabilization of band 3 to heat denaturation (half-maximal change in theta 222.5 = 61 degrees C). Circular dichroism studies of membranes that had been digested extensively with proteolytic enzymes and stripped of all extrinsic fragments revealed that the portions of red cell membrane proteins that are embedded in the lipid bilayer contain a very high (86-94%) content of alpha-helix.

Amino Acids↗
PubMed1985

Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbodiimide.

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.

4-Chloromercuribenzenesulfonate↗
PubMed1984

Inhibition of phosphate transport in human erythrocytes by water-soluble carbodiimides.

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.

Binding Sites↗
PubMed1984

Characterization of skeletal muscle calsequestrin by 1H NMR spectroscopy.

Calsequestrin (Mr = 44,000) is a calcium-binding (KD congruent to 1 mM, congruent to 50 sites/molecule) protein found in the lumen of the sarcoplasmic reticulum of skeletal muscle. The 1H NMR spectrum of calsequestrin in the calcium-free form is presented and is characteristic of a protein largely in the random coil configuration. A number of peaks in the aromatic region have been assigned based on their chemical shifts and sensitivity to pH. The interaction of this protein with Ca2+ and K+ was studied by 1H NMR. Potassium ion binding to calsequestrin caused broadening and concomitant loss of intensity in both the aromatic and aliphatic regions of the spectrum. Calcium ion binding caused similar effects but at much lower metal ion concentrations. It was found that the binding of Ca2+ to calsequestrin was cooperative (Hill coefficient n = 2.9 +/- 0.2) with a dissociation constant of 0.25 +/- 0.06 mM in the absence of K+. In contrast, K+ showed binding to a single class of independent sites (KD = 0.20 +/- 0.04 M). Calcium binding was also studied by circular dichroism at protein concentrations similar to the NMR experiments. The binding profile and cooperativity (n = 2.0 +/- 0.1, KD = 0.19 +/- 0.04 mM) were in agreement with the 1H NMR results. Circular dichroism studies performed at low protein concentrations to reduce the possible effect of calcium binding on the concentration of free calcium gave similar values of n = 2.42 +/- 0.14 and KD = 0.21 +/- 0.005 mM. This cooperativity was also observed in the presence of 100 mM KCl although the affinity for calcium has been significantly reduced (n = 1.65 +/- 0.09, KD = 0.87 +/- 0.036 mM). In view of the large number of calcium binding sites in calsequestrin, these small Hill coefficients show that calcium binding to calsequestrin is only mildly cooperative.

Animals↗
PubMed1984

Size and shape of rabbit skeletal muscle calsequestrin.

Calsequestrin, a calcium-binding protein isolated from rabbit skeletal muscle sarcoplasmic reticulum, was subjected to physiochemical analysis using sodium dodecyl sulfate gel electrophoresis, gel filtration, sedimentation, viscosity, and circular dichroism techniques. The effects of sodium dodecyl sulfate, alkaline pH, guanidine hydrochloride, and calcium ions on the hydrodynamic properties of the protein were studied. Calsequestrin, in the absence of calcium, had a random coil conformation with an alpha-helical content of 11%. Calsequestrin bound 1.7 mg of sodium dodecyl sulfate per mg of protein resulting in an increase in the alpha-helical content to 20%. The protein was completely random coil in guanidine hydrochloride and had a molecular weight of 42,000 as determined by gel filtration in the presence of this denaturant. Sedimentation equilibrium studies showed that calsequestrin was not subjected to aggregation and had a molecular weight of 38,000. Calsequestrin had a low sedimentation coefficient (2.20 S), a high Stokes radius (45 A), and a high intrinsic viscosity (27.1 ml/g) that increased slightly to 32 ml/g in the presence of guanidine hydrochloride, all indicative of a highly extended structure. Similar studies, performed at pH 9.5, revealed that the protein was even more asymmetric at alkaline pH. Calsequestrin bound 50 mol of calcium ions per mol of protein with an affinity of 1 mM as determined by gel filtration. Calcium binding was accompanied by a change of the protein from a highly extended structure (Rs = 45 A) to a much more compact structure (Rs = 35 A).

Animals↗
PubMed1984

Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes.

Band 3 (Mr = 95,000), the anion transport protein of human erythrocyte membranes exists primarily as a dimer in solutions of nonionic detergents such as octaethylene glycol mono-n-dodecyl ether (C12E8). The role of the oligomeric structure of Band 3 in the binding of [14C]4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), an inhibitor of anion transport (Ki = 1-2 microM), was studied by characterizing the interaction of BADS with dimers and monomers of Band 3 covalently attached to p-mercuribenzoate-Sepharose 4B. BADS bound to matrix-bound Band 3 dimers with an affinity of approximately 3 microM at a stoichiometry of 1 BADS molecule/Band 3 monomer, in agreement with the BADS binding characteristic of Band 3 in the membrane and in solutions of C12E8. Band 3 dimers could be attached to the matrix via one subunit by limiting the amount of p-chloromercuribenzoate on the Sepharose bead. Matrix-bound monomers were formed by dissociation of the dimers with dodecyl sulfate or guanidine hydrochloride. Complete removal of the denaturants allowed formation of refolded Band 3 monomers since the matrix-bound subunits could not reassociate. These refolded Band 3 monomers were unable to bind BADS. Release of the monomers from the matrix with 2-mercaptoethanol allowed reformation of dimers with recovery of the BADS binding sites. These results suggest that the dimeric structure of Band 3 is required for BADS binding and that the BADS binding sites may be at the interface between the two halves of the Band 3 dimer.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

Inhibition of anion transport in human red blood cells by 5,5'-dithiobis(2-nitrobenzoic acid).

The uptake of [32P]phosphate into human red blood cells was inhibited (Ki = 0.6 mM) by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor (Ki = 7 mM). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[14C]-benzamido-4'-aminostilbene-2,2'-disulfonate, a potent inhibitor of anion transport (Ki = 1-2 microM), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.

Anions↗
PubMed1981

The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane.

The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents, prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.

Amino Acid Sequence↗
PubMed1980

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin.

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.

Acetylation↗
PubMed1980

Ion pathways in proteins of the sarcoplasmic reticulum.

In summary, we have begun to characterize three different ion pathways in the sarcoplasmic reticulum. Ca2+-ionophoric activity has been traced to a 13,000-dalton CNBr fragment localized at the amino terminus of the ATPase molecule... The pathway involved in Ca2+ release can be distinguished from the pathway involved in Ca2+ uptake by its insensitivity to quercetin. An anion pathway is sensitive to DIDS and appears to be localized in the ATPase molecule

Amino Acid Sequence↗