Summary
Colloidal iron staining, calcium binding and enzyme activities were studied in the isolated rat heart sarcolemma. Colloidal iron staining of the sarcolemma revealed a high density of negatively charged sites associated with the cell surface. This membrane fraction was found to have calcium binding activity at both low (0.1 mM) and high (1.25 mM) concentrations of calcium. Pretreatment of the sarcolemma with either trypsin, phospholipase C or neuraminidase, was associated with a reduction in colloidal iron staining as well as decreased calcium-binding activity at high concentrations of calcium. Calcium binding at low concentrations was decreased by both trypsin and neuraminidase. Mg2+ ATPase, Ca2+ ATPase, and Na+−K+ ATPase activities were altered by neuraminidase and trypsin treatments, whereas phospholipase C treatment altered Na+−K+ ATPase only. It is concluded that both surface negative charge and calcium-binding sites associated with the isolated rat heart sarcolemma are contributed by a mosaic of biomolecules including proteins, phospholipids and glycoproteins, and alterations in the surface charge may influence the activities of membrane-bound enzymes.
Zusammenfassung
Beim isolierten Sarkolemm vom Rattenherzen wurde die Färbung mit kolloidalem Eisen, Ca-Bindung und Enzym-Aktivitäten untersucht. Die Färbung mit kolloidalem Eisen zeigte eine hohe Dichte negativ geladener Stellen an der Zelloberfläche. Es wurde festgestellt, daß diese Membranfraktion über Ca-Bindungsaktivität sowohl bei niedriger (0,1 mM) als auch bei hoher (1,25 mM) Ca-Konzentration verfügt.
Vorbehandlung des Sarkolemms mit Trypsin, Phospholipase C oder Neuraminidase führte sowohl zu einer Abnahme der Färbung mit kolloidalem Eisen als auch zu einer verminderten Bindungsaktivität für Calcium bei hohen Ca-Konzentrationen. Die Ca-Bindung bei niedrigen Konzentrationen wurde sowohl durch Trypsin als auch durch Neuraminidase vermindert. Die Aktivität der Mg2+-ATPase, Ca2+-ATPase und Na+−K+-ATPase wurde durch Neuraminidase und Trypsin-Behandlung verändert, während Behandlung mit Phospholipase C nur die Na+−K+-ATPase beeinflußte. Es ergibt sich die Schlußfolgerung, daß sowohl den negativen Oberflächenladungen als auch den Ca-Bindungsstellen des isolierten Sarkolemms vom Rattenherzen ein Mosaik von Biomolekülen zugrunde liegt, welches Proteine, Phospholipide und Glycoproteine enthält, und daß Änderungen der Oberflächenladung die Aktivität membrangebundener Enzyme beeinflussen können.
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Supported by a Grant from the Medical Research Council (MRC) of Canada.
Supported by MRC Studentship award.
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Matsukubo, M.P., Singal, P.K. & Dhalla, N.S. Negatively charged sites and calcium binding in the isolated rat heart sarcolemma* . Basic Res Cardiol 76, 16–28 (1981). https://doi.org/10.1007/BF01908160
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DOI: https://doi.org/10.1007/BF01908160