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Enzymatic properties, metal composition and SH-group reactivity of the light and heavy sarcoplasmic reticulum
1Central Research Laboratory, University Medical School, Debrecen, Hungary.
Abstract:
Light (LT) and heavy (TTC) microsomes were isolated from the fragmented sarcoplasmic reticulum (FSR) of rabbit skeletal muscle by sucrose gradient centrifugation. The amount of the protein components (ATPase, "feet proteins", calsequestrine) showed substantial differences between the light and heavy fractions. The amounts of calcium, magnesium and zinc were about 2-4 times higher in the TTC fraction, then those of the LT fraction. The activities of the Ca2+ + Mg2+ activated para-nitrophenyl-phosphatase and acetylcholin-esterase were (about 1.5 times) also higher in the TTC fraction compared to the LT fraction. The ratio of the Ca-transport vs. ATP was 2.1 in case of the LT and 0.6 in case of the TTC fraction. The number of titerable SH-groups of the LT fraction, measured in EGTA-medium containing Ca2+, was higher than those measured in the absence of Ca2+, while for the TTC fraction this number was higher when determined in the absence of Ca2+. We suppose that due to the higher amount of Ca2+ and Zn2+ in the TTC fraction those SH-groups which were present as Ca- or Zn-thiolates became titerable in EGTA-medium in the absence of Ca2+.
Insights
Heavy microsomes (TTC) from rabbit skeletal muscle sarcoplasmic reticulum contain more proteins, minerals like calcium and zinc, and enzyme activities than light microsomes (LT). This suggests distinct functional roles in calcium regulation.
Area of Science:
- Muscle Physiology
- Biochemistry
- Cellular Biology
Background:
- Sarcoplasmic reticulum (SR) is crucial for calcium ion (Ca2+) regulation in muscle cells.
- Microsomal fractions of SR, differing in density, may represent distinct functional or structural components.
Purpose of the Study:
- To biochemically characterize light (LT) and heavy (TTC) microsomes isolated from rabbit skeletal muscle SR.
- To investigate differences in protein composition, mineral content, enzyme activities, and thiol group titratability between LT and TTC fractions.
Main Methods:
- Isolation of LT and TTC microsomes from fragmented SR (FSR) using sucrose gradient centrifugation.
- Quantification of protein components (ATPase, "feet proteins", calsequestrine).
- Measurement of Ca2+, Mg2+, Zn2+ content, Ca2+ + Mg2+ activated para-nitrophenyl-phosphatase and acetylcholinesterase activities.
- Determination of titratable SH-groups in the presence and absence of Ca2+.
Main Results:
- TTC fractions exhibited higher amounts of proteins, Ca2+, Mg2+, and Zn2+ compared to LT fractions.
- Enzyme activities (Ca2+ + Mg2+ activated para-nitrophenyl-phosphatase, acetylcholinesterase) were approximately 1.5 times higher in TTC fractions.
- The Ca-transport vs. ATP ratio was significantly different: 2.1 for LT and 0.6 for TTC.
- Titratable SH-groups showed differential behavior related to Ca2+ presence, suggesting Ca2+ or Zn2+ binding in TTC fractions.
Conclusions:
- LT and TTC microsomes represent distinct populations within the fragmented SR with differing biochemical properties.
- The higher mineral and enzyme content in TTC fractions indicates a potential role in active calcium transport and storage.
- Differences in SH-group titratability suggest a role for Ca2+ and Zn2+ in modulating protein structure or function within the TTC fraction.