Arun K. Chakrabarti

Distribution of calcium activated neutral proteinase (mM CANP) in myelin and cytosolic fractions in bovine brain white matter

The activity of calcium-activated neutral proteinase (mM CANP) was determined in homogenate, myelin and supernatant of bovine brain corpus callosum. The enzyme activity in homogenate and myelin was increased eleven and thirteen-fold respectively by Triton X-100. Myelin prepared by the method of Norton and Poduslo as well as by a modified method, was shown to contain most (more than 50%) of homogenate mM CANP activity. The specific activity was highest in myelin, and increased almost three times more than the homogenate. Supernatant only contained 17% of enzyme activity. It is concluded from these studies that mM CANP is tightly bound to the membrane and predominantly associated with the myelin sheath.

Purification of Calcium-Activated Neutral Proteinase (CANP) From Purified Myelin of Bovine Brain White Matter

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN3, 5 mM β-mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 μg). It required 0.8 mM Ca2+ for half-maximal activation and 5 mM Ca2+ for optimal activation. Mg2+ (5 mM) was ineffective while Zn2+ and Hg2+ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.

Regulation of brain m calpain Ca2+ sensitivity by mixtures of membrane lipids: activation at intracellular Ca2+ level.

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 μM Ca2+ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca2+ level. GD1a (100 μM) and cerebroside (Cerb; 750 μM; 1:7.5) mixture was the most effective. At 0.5 μM to 1.0 Ca2+ concentrations, 15–20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 μM), GM1 (100 μM), Cerb (750 μM), sulfatide (Sulf; 750 μM), and phosphatidylinositol (PI; 300 μM) at a ratio of 1:1:7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three‐ to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 μM Ca2+ concentration. Triton X‐100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca2+ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca2+ concentration (0.4 μM), confirming the glycolipid‐mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca2+.

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P1 (nuclear), P2 (mitochondrial) and P3 (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P1A, P2A, P3A) which separate at the interphase of 0.32 M- and 0l85 M-sucrose. The distribution of activity was P2A>P1A>P3A. Further purification of myelin (of P2A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20–39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20–30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.

REGULATION OF THE CALCIUM-ACTIVATED NEUTRAL PROTEINASE (CANP) OF BOVINE BRAIN BY MYELIN LIPIDS

Since calcium-activated neutral proteinase (CANP; calpain) activation occurs at the plasmalemma and the enzyme is found in myelin, we examined myelin lipid activation of brain CANP. Purified lipids were dried, sonicated and incubated with purified myelin CANP. The CANP was assayed using [14C]azocasein as substrate and the Ca2+ concentration ranged from 2 microM for muCANP to 5 mM for mCANP. Phosphatidylinositol (PI), phosphatidylserine (PS) and dioleoylglycerol stimulated the mCANP activity by 193, 89 and 78%, respectively. PI stimulated both m- and muCANP in a concentration-dependent manner, while phosphatidylcholine was least effective. Cerebroside and sulfatide at higher concentrations (750 microM) were stimulatory. The phospholipid (PL)-mediated activation was inhibited by the PL-binding drug trifluoperazine. PI reduced the Ca2+ requirement for CANPs significantly (20-fold). These results suggest that acidic lipids and particularly acidic phospholipids activate membrane CANP.

Calcium-activated neutral proteinase (calpain) in rat brain during development: compartmentation and role in myelination

The activity of both forms (microM and mM Ca(2+)-sensitive) of calcium-activated neutral proteinase (calpain) was determined in developing rat brain. Triton X-100 did not affect mcalpain activity at the earlier ages (1-5 days postpartum) whereas mcalpain activity significantly increased at 16 days and older. The mcalpain activity in brain was negligible at earlier ages (1-7 days) and the peak activity occurred between 16 and 30 days after birth. The peak activity of mcalpain in myelin was found between 16 and 30 days of age and myelin from rats older than 30 days contained 40-50% of the brain mcalpain activity. In contrast, 70-80% of the brain mcalpain activity was in cytosol at younger ages (1-10 days) and decreased to 30% with increasing age (90 days). On the other hand, mu calpain was found mainly (65-75%) associated with a membrane fraction (microsomes) before 10 days and the majority of the activity was found in cytosol (68%) between 16 and 30 days. Immunoblot studies revealed mcalpain in both myelin and cytosol from developing rat brain. These results indicate that mcalpain is present in myelin and suggest that it may be involved in the formation of myelin sheath.

Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma‐enriched fraction, cAMP, or NGF showed concentration‐dependent inhibition of both μcalpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated μcalpain (37%) and mcalpain (58%) of tSc while PDGF‐aa and PDGF‐bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF‐aa, and PDGF‐bb compared to control while this activity was 40% with FGF‐treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain‐calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of μcalpain (37%) and mcalpain (36.5%) activity compared to treated cells from which the growth factors were not withdrawn. The μcalpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30–40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48–72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet‐derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDG‐Fbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non‐nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors. J. Neurosci. Res. 47:609–616, 1997.

Effects of detergents on Ca2+-activated neural proteinase activity (calpain) in neural and non-neural tissue: A comparative study

Calcium activated neutral proteinase (mcalpain) activity was determined in brain and other tissue of rat. More than 60% of the brain mcalpain activity was present in the particulate fraction while only 30% was in cytosol. In contrast, particulate fractions of liver, kidney, muscle, and heart contained about 8–12% of tissue mcalpain activity while 88% was present in cytosol. Removal of the endogenous inhibitor calpastatin increased the tissue mcalpain activity severalfold. Triton X-100 and deoxycholate (DOC) stimulated the neural calpain activity by ten-fold while activity in non-neural tissue was unaffected. Incubation with other detergents, e.g. Triton N-57 and thioglucopyranoside, stimulated brain calpain activity five-fold while Brij-35 did not have any effect. Sodiumdodecylsulphate (SDS), on the other hand, inhibited the enzyme activity. Brain contained the lowest calpain activity compared to non-neural tissue. The calpain activity in muscle, kidney and heart was three-fold greater than liver. Immunoblot identification of the enzyme revealed that calpain was predominantly in the particulate fraction and less in cytosol of brain while it was present mainly in cytosol and less in the pellet fractions of non-neural tissue.