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Related Experiment Videos

Differences in sarcolemmal preparations: cell surface material and membrane sidedness.

M P Moffat, P K Singal, N S Dhalla

    Basic Research in Cardiology
    |July 1, 1983
    PubMed
    Summary

    Two methods for isolating rat heart sarcolemma were compared. One method preserved cell surface material, yielding more realistic functional data for Na+-K+ ATPase and adenylate cyclase activities.

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    Area of Science:

    • Cardiovascular Biology
    • Membrane Biochemistry
    • Cellular Physiology

    Background:

    • Sarcolemma isolation is crucial for studying cardiac function.
    • Understanding sarcolemmal properties requires pure and representative membrane preparations.
    • Previous methods may not fully preserve native sarcolemmal characteristics.

    Purpose of the Study:

    • To compare two sarcolemma isolation methods: hypotonic shock-LiBr (HL) and sucrose density gradient (S).
    • To evaluate the impact of cell surface material and membrane sidedness on functional characteristics.
    • To determine which method yields sarcolemmal preparations most representative of in situ conditions.

    Main Methods:

    • Isolation of rat heart sarcolemma using hypotonic shock-LiBr (fraction HL) and sucrose density gradient (fraction S).

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  • Enrichment analysis of Na+-K+ ATPase and adenylate cyclase activities.
  • Assessment of cell surface material using cationic dyes, lanthanum, and ferritin.
  • Measurement of ATP-independent and ATP-dependent Ca2+ binding.
  • Evaluation of Ca2+-stimulated Mg2+-dependent ATPase activity.
  • Inhibition studies with ouabain on Na+-K+ ATPase.
  • Deoxycholate treatment to assess membrane sidedness effects.

    • Main Results:

    • Both methods enriched Na+-K+ ATPase and adenylate cyclase 8-13 fold.
    • Fraction HL showed cell surface material, high sialic acid, and 6x more ATP-independent Ca2+ binding.
    • Fraction S had less sialic acid, no cell surface staining, and 4-6x higher ATP-dependent Ca2+ binding and Ca2+-stimulated Mg2+-dependent ATPase.
    • Epinephrine stimulated adenylate cyclase more in HL (24%) than S (3%).
    • Ouabain inhibited Na+-K+ ATPase significantly in HL (80%) vs. S (10%), with near-complete inhibition after deoxycholate treatment in both.

    Conclusions:

    • Cell surface material and membrane sidedness significantly influence the in vitro functional characteristics of sarcolemma.
    • Sarcolemmal preparations retaining cell surface material (Fraction HL) provide more physiologically relevant data.
    • Methodological choices in sarcolemma isolation impact the interpretation of enzyme activities and ion binding.