Regulatory differences among avian ecto-ATPases.

Extracellular ATP is found to produce a variety of important biological responses. Ecto-ATPases are located on numerous cell types in many different species, regulate extracellular ATP levels and can be a key step in generating adenosine. Studies conducted on chicken ecto-ATPases from liver and cardiac and smooth muscle show a variety of differing properties including (1) different apparent Km's, (2) lectin sensitivity, (3) responses to detergents, (4) responses to lipid mediators, and (5) responsiveness to nucleotide-mimetic affinity labels. These results suggest that although each enzyme hydrolyzes extracellular ATP, they should each be viewed as a distinct subtype of the whole ecto-ATPase family due to their differential responses, largely linked to proposed regulatory phenomenon.

The skeletal muscle transverse tubular Mg-ATPase: identity with Mg-ATPases of smooth muscle and brain.

Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M(r) 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M(r) 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P1- and P2-purinoceptors. It is also proposed that the ecto-MgATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-MgATPase may modulate the neurotransmitter role of ATP.

Direct effects of phorbol esters and diacylglycerols on the T-tubule Mg(2+)-ATPase.

T-tubule membrane vesicles isolated from skeletal muscle contain a very active Mg(2+)-ATPase (EC 3.6.1.34) which is modulated by lectins and is located in the junctional region near the sarcoplasmic reticulum membranes (1). The effects of several prominent lipophilic agents upon the ATPase have led us to evaluate the action of diacylglycerols and phorbol esters upon the enzyme. The ATPase is inhibited by submicromolar levels of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (sn-OAG), with K0.5s of 0.2 and 0.5 microM, respectively. Significantly, 4-alpha-phorbol 12,13-didecanoate (4-alpha-phorbol) the TPA analogue shown to be inactive toward protein kinase C (PKC), inhibited the ATPase with a K0.5 of 0.3 microM, and 1-stearoyl-2-arachidonyl-sn-glycerol, the preferred endogenous activator of PKC, was not inhibitory toward the ATPase. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (a membrane permeant PKC inhibitor) and peptide 19-36 (the highly specific PKC pseudosubstrate inhibitor) were both without effect upon the ATPase and did not affect TPA inhibition. ATPase activity was not altered under phosphorylating conditions in experiments using exogenous rat brain PKC. ConA protected ATPase activity against inhibition by TPA, 4-alpha-phorbol, and sn-OAG. Additionally, phorbol-12,13-dibutyrate binding studies demonstrated that the ATPase was capable of significant phorbol binding with ConA protection. The data are consistent with a direct and specific effect of phorbol esters and diacylglycerols upon the ATPase, without any participation of PKC. We conclude that the transverse tubule (T-tubule) ATPase is an alternate receptor for diacylglycerol and TPA in skeletal muscle and that the mode of action of these agents upon the ATPase (inhibition) is opposite to their mode of action on PKC (activation). The data demonstrate that substantial care must be taken in ascribing either cellular or subcellular effects of phorbol esters and diacylglycerols exclusively to the activation of PKC and that alternate receptors may exist. Criteria are recommended for the demonstration of PKC-independent modulation by phorbols and diacylglycerols.

Amino acid sequence of chicken calsequestrin deduced from cDNA: comparison of calsequestrin and aspartactin.

We have previously reported the amino terminal sequence of adult chicken calsequestrin, an intraluminal Ca2(+)-binding protein isolated from fast-twitch skeletal muscle. The partial sequence showed homology with mammalian calsequestrins contained in the PIR data bank and complete identity with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin. Based on these data, oligonucleotide primers were synthesized for PCR amplification and direct DNA sequencing. We report herein the primary sequence of chicken calsequestrin, deduced from cDNA. The sequence has been verified by amino acid sequencing of internal tryptic peptides. Importantly, the data show the primary structure of calsequestrin to be identical to the amino acid sequence reported for aspartactin, with the exception of a single amino acid difference (ileu vs. val) which may be animal strain-related. Based on these data, calsequestrin and aspartactin are the same protein.

Calsequestrin, an intracellular calcium-binding protein of skeletal muscle sarcoplasmic reticulum, is homologous to aspartactin, a putative laminin-binding protein of the extracellular matrix.

Calsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed.

Biochemical properties of isolated transverse tubular membranes.

This review addresses the major biochemical and structural characteristics of isolated transverse tubule (T-tubule) membranes, including methods of isolation and morphology of purified membranes, evaluation of attendant membrane activities, including ion pumps and channels, and structural and compositional analyses of functionally relevant components. Particular emphasis is placed on the Mg2+-ATPase, its localization in the T-system, its unusual kinetic properties, its possible functions, and its potential regulation by diacylglycerol and other biologically-relevant lipids. Conclusions are drawn with respect to the biochemical markers characteristic of T-tubule membranes and the criteria to be applied in the assessment of isolated T-tubule membrane purity.

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase.

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.

Studies on the transverse tubule membrane Mg-ATPase. Lectin-induced alterations of kinetic behavior.

Transverse tubule (TT) membrane vesicles contain a very active Mg-ATPase (EC 3.6.1.3). Concanavalin A (ConA) and other lectins were found to activate the TT Mg-ATPase from chicken skeletal muscle up to 25-fold yielding specific activities greater than 800 mumol/h/mg. The sarcoplasmic reticulum Ca-ATPase and the sarcolemma Na,K-ATPase were unaffected by ConA. 125I-Labeled lectin binding to the TT membrane Mr 102,000 glycoprotein supports the contention that this protein is identical with or is intimately associated with the TT Mg-ATPase. The ATPase exhibited non-Michaelis-Menton kinetics with both apparent negative cooperativity (n = 0.723; S0.5, Mg-ATP = 14 microM) and substrate inhibition (Ki, Mg-ATP = 10.2 mM), both of which were eliminated in the presence of ConA. Under the same conditions, ConA also abolished the unusual temperature dependence and potent Triton X-100 inhibition. The similarities in ConA suppression of both Triton and substrate inhibition suggest that these ligands may be interacting through a non-catalytic site and that Triton is serving as a nucleotide-mimetic agent. The unique kinetic responses are consistent with a homotropic substrate modifier mechanism wherein the enzyme can be viewed as possessing a single catalytic and a single regulatory site on a single polypeptide chain. It is proposed that ConA interferes either with ligand interaction at a putative regulatory site or blocks communication between a regulatory site and the catalytic site. The possible nature of the regulatory site and its modulation by a ConA-like, endogenous, skeletal muscle lectin and their combined role in excitation-contraction coupling is discussed.

Characterization of transverse tubule membrane proteins: tentative identification of the Mg-ATPase.

Vesiculated fragments of chicken skeletal muscle transverse tubule (TT) membranes were analyzed for their content of loosely associated and integral membrane proteins. Of particular interest was the identification of the magnesium-stimulated ATPase (Mg-ATPase), which is characteristically located in native isolated TT vesicles of chicken skeletal muscle [R. A. Sabbadini and V. R. Okamoto (1983) Arch. Biochem. Biophys. 223, 107-119]. A number of the proteins found in vesicular TT preparations were found to be extractable by a mild Triton-X100 treatment and were identified as aldolase, enolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and pyruvate kinase. Approximately 60% of TT-associated protein was extracted with Triton, resulting in a twofold enrichment of the Mg-ATPase. Concommitantly, one core integral membrane protein possessing a Mr of 102,000 was enriched, suggesting that it is responsible for the Mg-ATPase activity present in chicken skeletal muscle TT membranes.

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives.

Fluorescent 3'-O-acyl-substituted adenine nucleotide (dimethylamino)naphthoyl and trinitrophenyl groups were studied for binding to the ADP/ATP carrier in mitochondria and submitochondrial particles. The changes in fluorescence intensity and emission maximum are for the most part similar to those observed in nonaqueous solvents. The (dimethylamino)naphthoyl derivatives from a largely quenched aqueous state have a shortwave shift up to 85 nm and increase up to 90-fold (1,5 derivative), whereas the little quenched naphthoyl derivatives show a fluorescence decrease and the weakly fluorescent trinitrophenyl derivative shows only a small increase on binding. All derivatives are good inhibitors (K1 = 1-10 microM) of nucleotide transport. The fluorescence titrations have an apparent K1/2 = 2-7 microM. The fluorescence of the 1,5-DAN nucleotide is fully suppressed by bongkrekate but only partially suppressed by carboxyatractylate. The fluorescence response is much stronger in submitochondrial particles than in mitochondria. Both facts suggest fluorescent binding to the "m" state of the carrier site at the inner face of the membrane. 1,5-DAN-AMP shows a slightly more efficient binding than DAN-ADP or DAN-ATP.

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2. [5-(Dimethylamino)-1-naphthoyl]adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier.

The binding to the ADP/ATP carrier in mitochondrial membranes of the 3'-O-(dimethylamino)naphthoyl (DAN) derivatives of AMP, ADP, and ATP was quantitatively analyzed. The sidedness of the fluorescent type binding to the "m" side only was shown comparing the mitochondrial membranes in various stages of integrity and surface orientation. In particles displacement by bongkrekate (BKA) is direct, whereas in the case of carboxyatractylate (CAT) the requirement for ADP and ATP demonstrates the transition from the "m" to the "c" state. Quantitatively the "physical" binding of [3H]DAN-AMP and fluorescence are well correlated, allowing for a little nonfluorescent binding to the c side. For DAN-AMP KD is 1.6 microM, for DAN-ADP KD is 0.8 microM, and in the Hill plot a straight line with n = 1.25 is obtained. The maximum number of binding sites for [3H]DAN-AMP (1.5 mumol/g of protein) is about equal to the sites found for [3H]BKA if the unspecific binding of both ligands is differentiated by blocking carrier sites with CAT. [3H]CAT binding is somewhat lower in accordance with the limited access of CAT to inverted vesicles. ADP is able to decrease fluorescence only by about 35% at high concentrations (10 mM) whereas GDP has virtually no effect. With ADP, DAN-AMP binding decreases by 30% of the total binding sensitive to BKA. Binding to ATPase is low because of the absence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)