Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles.
Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.
Kim
D H
DH
Department of Muscle Research, Boston Biomedical Research Institute, MA.
Sreter
F A
FA
Ikemoto
N
N
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D008305:Malignant Hyperthermia / Q000503:physiopathology*; D009124:Muscle Proteins / Q000502:physiology*; D009132:Muscles / Q000503:physiopathology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine
D009124:Muscle Proteins; D010750:Phosphoproteins; D002118:Calcium