Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses

Marjorie Shih, Larry David, Kirsten Lampi, Hong Ma, Chiho Fukiage, Mitsuyoshi Azuma, Thomas (Tom) Shearer

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, βH crystallin, or recombinant βB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human βB1, βA3, and βB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of α- and β-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

Original languageEnglish (US)
Pages (from-to)458-469
Number of pages12
JournalCurrent Eye Research
Volume22
Issue number6
DOIs
StatePublished - 2001

Fingerprint

Crystallins
Lenses
Proteolysis
Light
Hot Temperature
Calpain
m-calpain
In Vitro Techniques
Cataract
Electrophoresis
Polyacrylamide Gel Electrophoresis
Rodentia
Peptides

Keywords

  • Calpain
  • Human and bovine lenses
  • Lens crystallins
  • Light scattering
  • Proteolysis

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses. / Shih, Marjorie; David, Larry; Lampi, Kirsten; Ma, Hong; Fukiage, Chiho; Azuma, Mitsuyoshi; Shearer, Thomas (Tom).

In: Current Eye Research, Vol. 22, No. 6, 2001, p. 458-469.

Research output: Contribution to journalArticle

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abstract = "Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, βH crystallin, or recombinant βB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human βB1, βA3, and βB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of α- and β-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.",
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AU - Shih, Marjorie

AU - David, Larry

AU - Lampi, Kirsten

AU - Ma, Hong

AU - Fukiage, Chiho

AU - Azuma, Mitsuyoshi

AU - Shearer, Thomas (Tom)

PY - 2001

Y1 - 2001

N2 - Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, βH crystallin, or recombinant βB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human βB1, βA3, and βB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of α- and β-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

AB - Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, βH crystallin, or recombinant βB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human βB1, βA3, and βB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of α- and β-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

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KW - Proteolysis

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