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H B Cunningham

Publications and source records attributed to H B Cunningham.

4 recordsLinked to original sources
PubMed1993

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.

Animals↗
PubMed1991

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.

Animals↗