Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples

Clark, Arthur; DeMartino, George N.; Croall, Dorothy E.

doi:10.1042/bj2350279

Cited by 22 publications

(5 citation statements)

References 16 publications

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“…On the other hand, for all the nutritional conditions studied, the sum of the three DEAE-Sephacel-eluted activities (total calpain activity) was higher than the activity displayed by the corresponding soluble homogenate. This is in keeping with results of Clark, De Martino and Croall (1986) and indicates that calpains were separated from calpastatin as has been described previously (Murachi 1983). In addition, the complete homogenate from depleted liver displayed a proportion of its total calpain activity higher compared to normal and refed one and, consequently, suggested that the content of calpastatin decreased during protein depletion and increased during refeeding.…”

Section: Discussionsupporting

confidence: 91%

Effect of Dietary Level of Protein on the Activity of Mouse Liver Calpain

Goicoechea

Tabares²,

Sabas³

et al. 1994

Horm Metab Res

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The effect of protein depletion and refeeding with a normal diet on mouse liver soluble homogenate calpain activity was studied. It was unchanged when expressed in terms of whole liver (units/liver). However, when expressed in terms of degradable protein (units/mg protein) it increased with depletion and decreased with refeeding. DEAE Sephacel chromatography of soluble homogenate yielded three calpain activities which were eluted at 0.04, 0.16 and 0.23 M NaCl, respectively. On the basis of whole liver, they decreased with depletion and increased with refeeding. Immunochemical analysis revealed similar changes in the mass of the calpain eluted with 0.16 M NaCl. The sum of these three activities (total liver calpain activity) was higher than the activity displayed by the soluble homogenate, indicating that they were separated from calpastatin. Furthermore, the percentage of total calpain activity displayed by soluble homogenate increased with depletion and decreased with refeeding, suggesting that depleted liver had the lowest calpastatin content. This was confirmed by direct measurements which indicated that depleted homogenate had in average 5.5 and 3.1 times less calpastatin compared to normal and 16 hours refed liver, respectively. It is concluded that a remarkable decrease in calpastatin content maintained unchanged whole liver soluble homogenate calpain activity during protein depletion and refeeding and contributes to an increased calpain activity related to degradable protein in depleted livers. This increase is in accordance with the high in vivo rate of protein breakdown depicted by these livers.

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Section: Discussionsupporting

confidence: 91%

Effect of Dietary Level of Protein on the Activity of Mouse Liver Calpain

Goicoechea

Tabares²,

Sabas³

et al. 1994

Horm Metab Res

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“…Although immunoreactive content directly reflected activity after mCANP was partially purified by ion-exchange chromatography in this study, activity measurements are often subject to too many influences to be an accurate index of mCANP content. mCANP activity, for example, is reported to be modulated by protein co-factors (calmodulin, endogenous protein activators and inhibitors) and other cellular constituents (phospholipids, polyamines, complex lipids, gangliosides) (Murakami et al, 1981;Clark et al, 1986;Gopalakrishna and Barsky, 1986). Immunoassay, by avoiding these influences, measures the actual amount of mCANP protein in a preparation.…”

Section: Discussionmentioning

confidence: 99%

Immunoassay and Activity of Calcium‐Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane‐Associated Fractions in Human and Mouse Brain

Takeuchi

Saitô

Nixon

1992

Journal of Neurochemistry

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The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.

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“…Calpain Activity Assay. Supernatant from the tissue homogenate (50-70 mg/ml, 2 ml) was chromatographed on a Reactive Red-agarose column (0.8 £ 2 cm) to separate calpain from calpastatin in a single step (13). The proteolytic activity of the chromatographed fractions was determined by using the uorogenic peptide Suc-Leu-Tyr-7-amino-4-methylcoumarin (Calbiochem, San Diego CA) as substrate (14).…”

Section: Methodsmentioning

confidence: 99%

The Calpain Small Subunit Gene Is Essential: Its Inactivation Results in Embryonic Lethality

Zimmerman

2000

IUBMB Life

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Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.

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Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples

Cited by 22 publications

References 16 publications

Effect of Dietary Level of Protein on the Activity of Mouse Liver Calpain

Effect of Dietary Level of Protein on the Activity of Mouse Liver Calpain

Immunoassay and Activity of Calcium‐Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane‐Associated Fractions in Human and Mouse Brain

The Calpain Small Subunit Gene Is Essential: Its Inactivation Results in Embryonic Lethality

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