Association of calpain with insoluble pellet of rat lens

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.

Original languageEnglish (US)
Pages (from-to)369-375
Number of pages7
JournalExperimental Eye Research
Volume55
Issue number2
DOIs
StatePublished - 1992

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Calpain
Lenses
Calcium
Membranes
Water
Sucrose
Urea

Keywords

  • calcium
  • calpain
  • calpastatin
  • lens
  • membrane

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Association of calpain with insoluble pellet of rat lens. / Lampi, Kirsten; David, Larry; Shearer, Thomas (Tom).

In: Experimental Eye Research, Vol. 55, No. 2, 1992, p. 369-375.

Research output: Contribution to journalArticle

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abstract = "Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10{\%} was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30{\%} of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.",
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AB - Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.

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