Phosphorylase Kinase Isoenzymes in Deficient ICR/IAn Mice

Abstract

ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues (Lyon, S. B. Biochem. Genet. 4, 169–185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P. T. W. & Cohen, P. FEBS Lett. 29, 113–115 (1973)]. New‐born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity. The properties of these residual phosphorylase kinases were compared to those of control C57BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen‐Proux et al. Biochim. Biophys. Acta, 452, 398–405 (1976)]. The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new‐born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme. These results lead to the conclusion that there exists in the muscle of I strain a ‘foetal form’ of phosphorylase kinase, the activity of which decreases progressively after birth. Our work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347–356 (1976)] which showed that there is no evidence for the existence of a cross‐reacting material in the muscle of adult deficient mice.

Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice.

“…There is considerable precedence for separate cardiac and skeletal muscle isoforms of proteins being present at various levels in both muscle types, examples of which include creatine kinase and a-actin (14,35,36). Evidence supporting this possibility has been demonstrated (6) by the observation that cardiac muscle Phk has properties similar to neonatal skeletal muscle Phk, which are distinct from the properties of the adult skeletal muscle isoform. It has also been shown that Phk activity in neonatal I-strain skeletal muscle is not reduced to nearly the extent found in adult I-strain skeletal muscle (4,6).…”

“…Evidence supporting this possibility has been demonstrated (6) by the observation that cardiac muscle Phk has properties similar to neonatal skeletal muscle Phk, which are distinct from the properties of the adult skeletal muscle isoform. It has also been shown that Phk activity in neonatal I-strain skeletal muscle is not reduced to nearly the extent found in adult I-strain skeletal muscle (4,6). The y-Phk mRNA species observed in cardiac muscle and brain (Fig.…”

“…In addition to the almost complete lack of Phk protein or activity in I-strain skeletal muscle, there is a partial reduction of Phk activity in I-strain cardiac muscle and brain when compared to control mice (4,6). It is, therefore, of interest that cardiac muscle and brain also contain hybridizable y-Phk mRNAs the same size as found in skeletal muscle.…”

“…It has been shown that the isolated y subunit possesses considerable enzymatic activity both by itself and when complexed with the 8 subunit calmodulin (28,29). The observation that I-strain muscle contains <0.2% of the control levels of Phk activity argues that functional 'y subunit is not present at an appreciable concentration (4,6,12). Furthermore, no Phk protein is detectable in I-strain skeletal muscle via standard purification protocols or immunoprecipitation techniques (12).…”

“…Skeletal muscle contains the highest amounts of Phk enzymatic activity, although activity is also observed in liver, cardiac muscle, brain, and several other tissues (4). Kinetic differences exist between the cardiac and adult skeletal muscle enzymes, and the liver and skeletal muscle enzymes are known to be separate isozymes (5)(6)(7). Cardiac muscle and red skeletal muscle Phk contain an isoform of the a subunit, termed a', distinct from that found in white skeletal muscle (8).…”