Effect of alloxan diabetes on a Ca2+-activated proteinase in rat skeletal muscle.

The effect of making rats diabetic by alloxan injection on activity of the muscle Ca2+-activated proteinase (CAF) was investigated. Groups of four to seven control or alloxan-injected rats were killed 10 min (0 day) and 10, 17, and 24 days after a second alloxan injection. The second alloxan injection was given 3 days after the first. CAF activity was assayed in fractions precipitated between 0 and 45% ammonium sulfate saturation (P0-45 crude CAF fractions) that had been prepared so as to remove the protein inhibitor of CAF. Gel permeation, followed by DEAE-cellulose chromatography of pooled P0-45 crude CAF fractions from each time-treatment group, demonstrated that the assays used in this study were specific for CAF activity. Muscle CAF activity was up to 50% higher in alloxan-injected rats than in control rats, regardless of whether activity was expressed per gram sarcoplasmic protein, per gram contractile protein, or per gram skeletal muscle fresh weight. Alloxan injection diminished rate of muscle mass accumulation but did not change the proportion of sarcoplasmic or contractile protein in skeletal muscle. Hence, alloxan injection decreased the rate of contractile protein deposition. The elevation of muscle CAF activity in alloxan-injected rats is consistent with the proposed role of CAF in initiating metabolic turnover of myofibrillar proteins but does not prove this role nor exclude participation of other proteinases.

Effect of starvation and refeeding on activity of a Ca2+-dependent protease in rat skeletal muscle.

The effects of starving, refeeding, and restarving rats for different periods on content of sarcoplasmic and contractile proteins and on activity of the Ca2+-dependent proteinase (CAF) in skeletal muscle was determined. Groups of five to six male rats, 8 to 11 weeks old, were starved up to 8 days, refed up to 6 days, and in two experiments, restarved up to 10 days. CAF activity was assayed in P 0-45 crude CAF fractions prepared so as to remove a protein inhibitor of CAF; the assays were demonstrated to be specific for CAF. Sarcoplasmic protein content of rat skeletal muscle changed little until after 6 days of restarvation when it decreased to 68-89% of control level (P less than 0.05). Contractile protein content decreased to 65% of control level (P less than 0.01) after 8 days of starvation, remained at this level for 4 days of refeeding, then increased to approximately 80% of control level after 6 days of refeeding, remained at this level for 2 days of restarvation, and then decreased to approximately 65% of control level (P less than 0.01) after 6 and 8 days of restarvation. Muscle CAF activity did not change during the first 8 days of starvation but increased to 113% above control level (P less than 0.01) after 6 days refeeding and then decreased to only 29% of control level (P less than 0.01) after 8 days of restarvation. These changes in muscle CAF activity are consistent with the proposed role for CAF in initiating metabolic turnover of contractile proteins, but because actual measurements of myofibrillar protein were not made and because in vivo CAF activity is difficult to assess, they do not prove this role for CAF nor do they exclude participation of other proteinases.

Degradation of myofibrillar proteins by trypsin-like serine proteinases.

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

Proteolysis of myofibrillar proteins at neutral pH.

Myofibrils that had been exposed to rat pancreatic trypsin-like serine proteinase and to beef heart Ca2+-activated thiol proteinase were examined in the electron microscope and by SDS-gel electrophoresis. The former enzyme caused more extensive disruption of the ultrastructure and degraded more of the myofibril proteins than the CA2+-activated proteinase. The susceptibilities of individual purified proteins to the two enzymes were also compared. Myosin was virtually resistant to the Ca2+-activated enzyme but with smooth muscle myosin/rat serine proteinase at a ratio of 20000/1, heavy chain degradation took place very rapidly and the ability of the degraded myosin to have its ATPase activity activated by actin in the presence of Ca2+ was lost at a similar rate. G-actin, troponins T and I and alpha-actinin were also degraded readily by the trypsin-like proteinase whereas tropomyosin, a negatively charged rodlike protein, was more resistant. The cellular location of both proteinases remains to be established but from these results obtained in vitro, consideration is given to whether these types of proteinase might work cooperatively in vivo to bring about the disassembly and turnover of myofibrillar proteins that is known to take place outside the lysosomes in muscle.