Importance of monitoring calcium & calcium related properties in carrier detection for Duchenne muscular dystrophy.

Calcium and calcium dependent enzymes viz., calcium ATPase, protein kinase C and calcium activated neutral protease (milli CANP mCANP) were studied in the erythrocytes, platelets and lymphocytes of obligate carriers, in order to assess the usefulness of these indices for detection of carriers for Duchenne muscular dystrophy (DMD). With the exception of mCANP and lymphocyte calcium ATPase, other calcium dependent enzyme activities showed considerable overlap between carriers and control. Since the increase in the level of platelet mCANP was found in all affected boys (no false negatives) and obligate carriers, and patients with other myopathic conditions and some neurogenic causes did not show high platelet mCANP activity, this parameter could be considered as a good phenotypic index. Unlike SCK, the platelet mCANP of carriers did not overlap that of controls, hence tests are to be carried out to verify its usefulness as an index of carrier state in mutations other than DNA deletion since testing of non-deletion is both costly and has practical limitations.

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation.

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca 2+ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca2+ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.

Calcium activated neutral protease in blood cells from patients & carriers of Duchenne muscular dystrophy.

As blood cells such as platelets, lymphocytes and erythrocytes from patients with Duchenne muscular dystrophy show evidence of membrane alterations and elevation of intra-cellular calcium, one of the calcium related changes i.e., the activity of calcium activated neutral protease (CANP) was monitored and found to be elevated in erythrocytes, lymphocytes and platelets. As similar changes were observed in platelets of carriers of this disease, CANP in platelets may serve as a useful index for carrier detection.