Related Experiment Videos
ADP-ribosylation of Ca2+-dependent ATPase in vitro suppresses the enzyme activity
N Hara1, M Tsuchiya, K Mishima
1Central Research Laboratories, Shimane Medical University, Izumo, Japan.
Abstract:
We investigated the effect on the Ca2+-dependent ATPase activity of ADP-ribosylation of the enzyme from the rabbit skeletal muscle sarcoplasmic reticulum. A reconstituted ADP-ribosylation system of Ca2+-dependent ATPase in which the enzyme and ADP-ribosyltransferase, both were partially purified from the vesicles, and poly L-lysine were contained, was preincubated with 1 mM NAD, and the Ca2+-dependent ATPase activity was assayed. The NAD-dependent suppression of the enzyme activity depended on both the concentration of NAD and preincubation-time for the ADP-ribosylation, and was reversed by adding 20 mM arginine during the preincubation. These results taken together with the findings that Ca2+-dependent ATPase is a major acceptor protein for the modification in rabbit skeletal muscle sarcoplasmic reticulum [Hara et al. (1987) Biochem. Biophys. Res. Commun. 144; 856-862] suggest that Ca2+-transport in the sarcoplasmic reticulum may be regulated through changes in the rate of ADP-ribosylation of Ca2+-dependent ATPase.
Insights
ADP-ribosylation of calcium-dependent ATPase in rabbit skeletal muscle sarcoplasmic reticulum suppresses enzyme activity. This NAD-dependent regulation of calcium transport may be reversed by arginine.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Physiology
Background:
- Calcium ions (Ca2+) are crucial for muscle contraction, regulated by sarcoplasmic reticulum.
- Ca2+-dependent ATPase is the primary protein responsible for Ca2+ transport in the sarcoplasmic reticulum.
- ADP-ribosylation is a post-translational modification that can alter protein function.
Purpose of the Study:
- To investigate the effect of ADP-ribosylation on the Ca2+-dependent ATPase activity.
- To explore the regulatory role of ADP-ribosylation in Ca2+ transport within the sarcoplasmic reticulum.
Main Methods:
- A reconstituted system using partially purified Ca2+-dependent ATPase and ADP-ribosyltransferase from rabbit skeletal muscle sarcoplasmic reticulum.
- Preincubation with nicotinamide adenine dinucleotide (NAD) to induce ADP-ribosylation.
- Assay of Ca2+-dependent ATPase activity.
- Reversal experiments using arginine.
Main Results:
- NAD-dependent ADP-ribosylation significantly suppressed Ca2+-dependent ATPase activity.
- The suppression was dependent on both NAD concentration and preincubation time.
- The inhibitory effect of ADP-ribosylation was reversed by the addition of arginine.
Conclusions:
- Ca2+-dependent ATPase is a major target for ADP-ribosylation in skeletal muscle sarcoplasmic reticulum.
- ADP-ribosylation of Ca2+-dependent ATPase can regulate Ca2+ transport.
- This modification pathway offers a potential mechanism for controlling Ca2+ homeostasis in muscle cells.