Chicken skeletal muscle has three Ca2+-dependent proteinases

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Chicken breast muscle contains calcium-dependent proteinases (calpains) essential for various cellular processes.
• Two known calpains, mu-calpain (micromolar Ca2+ dependent) and m-calpain (millimolar Ca2+ dependent), are well-characterized in skeletal muscle.
• The existence and characteristics of other calpain forms in avian skeletal muscle remain less understood.

Purpose of the Study:

• To purify and characterize novel calcium-dependent proteinases from chicken breast muscle.
• To compare the properties of newly identified calpains with known mu- and m-calpains from other species.
• To investigate the potential existence of a high millimolar calcium-dependent calpain (high m-calpain) in chicken skeletal muscle.

Main Methods:

• Differential protein purification using various chromatography techniques including DEAE-cellulose, phenyl-Sepharose, octylamine agarose, Sephacryl S-300, and HPLC.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for polypeptide analysis.
• Western blotting using anti-calpain antibodies.
• Enzyme activity assays to determine Ca2+ dependency, pH optimum, and substrate specificity.
• Inhibition studies with calpastatin and other proteinase inhibitors.

Main Results:

• Three distinct Ca2+-dependent proteinases were identified: mu-calpain, m-calpain, and a novel high m-calpain.
• High m-calpain co-purified with mu-calpain but exhibited different elution profiles on DEAE-cellulose.
• Purified high m-calpain showed a unique high Ca2+ requirement (3800 microM) for maximal activity, distinct from mu-calpain (5.35 microM) and m-calpain (420 microM).
• High m-calpain shares functional similarities with other calpains, including limited myofibril digestion, pH optimum, Ca2+ requirement, and inhibitor sensitivity.
• Western blot analysis confirmed a 74-76 kDa polypeptide in high m-calpain preparations reacts with anti-calpain antibodies.

Conclusions:

• Chicken breast muscle harbors at least three distinct Ca2+-dependent proteinases, including a novel high m-calpain.
• High m-calpain represents a new class of calpain with significantly higher Ca2+ requirements than previously identified forms.
• The findings suggest that all skeletal muscles may contain both mu- and m-calpains, with varying proportions, and potentially novel high Ca2+-dependent variants.
• The physiological role of high m-calpain in chicken muscle warrants further investigation.