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J Verbist

Publications and source records attributed to J Verbist.

26 records · Page 2Linked to original sources

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Animals↗

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.

Adenosine Triphosphate↗

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

Animals↗

Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase.

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.

Animals↗

Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle.

The Ca2+ -transport ATPase [Ca2+ + Mg2+)-ATPase) in a plasma membrane-rich fraction of porcine antrum (stomach) smooth muscle, is stimulated 2.9-times by calmodulin in the presence of 0.2 mg/ml saponin and reaches a value of 12.0 +/- 2.0 (4) mumol/100 mg protein (equivalent to 110 g wet tissue) per min at 37 degrees C and 10(-5) M [Ca2+]. Saponin was found to specifically potentiate the calmodulin-(Ca2+ + Mg2+)-ATPase interaction, even in the Triton X-100 solubilized enzyme. The conditions for purification of the (Ca2+ + Mg2+)-ATPase by affinity chromatography on a calmodulin-Sepharose 4B gel were optimized. The purified enzyme has a specific activity of 11.9 mumol/mg protein per min at 37 degrees C, 10(-5) M [Ca2+], 0.6 microM calmodulin, and shows a double polypeptide band at 140 and 150 kDa. The (Ca2+ + Mg2+)-ATPase can be incorporated in artificial liposomes that thereupon show an ATP-dependent Ca2+ uptake (Ca:ATP = 1.0). The magnitude of the calmodulin stimulation of the isolated enzyme depends on its phospholipid environment. When isolated in the presence of phosphatidylserine no calmodulin stimulation is observed. After reconstitution in phosphatidylcholine the calmodulin stimulation amounts to 4.05 +/- 0.63 (n = 12) times.

Animals↗

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle.

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.

Animals↗

Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle.

Antibodies were raised against a calmodulin-binding CaMg-ATPase (Ca2+-transport ATPase) from smooth muscle. The binding of these antibodies to a number of related Ca2+-transport ATPases was studied. Antibodies to the calmodulin-binding ATPase from porcine antrum (stomach) smooth muscle do not only bind to this CaMg-ATPase, but also to the corresponding enzyme in porcine erythrocytes. However, they do not bind to the CaMg-ATPase from sarcoplasmic reticulum of porcine skeletal muscle. The binding of these antibodies to the CaMg-ATPase of smooth muscle, does not inhibit the enzyme activity.

Animals↗

Endoleaks after endoluminal repair of abdominal aortic aneurysms: diagnosis and treatment.

Endoleaks currently present one of the major problems related to endoluminal treatment of abdominal aortic aneuryms (AAA). The incidence and fate of endoleaks stress the importance of long term follow-up. In this article the authors discuss which imaging techniques can best be used for surveillance and how endoleaks should be classified. Furthermore the necessity of treatment as well as treatment options are discussed.

Aortic Aneurysm, Abdominal↗