Purification of calpain II from rat lens and determination of endogenous substrates

Larry L. David; Thomas R. Shearer

doi:10.1016/0014-4835(86)90057-6

Purification of calpain II from rat lens and determination of endogenous substrates

Larry L. David, Thomas R. Shearer

Research output: Contribution to journal › Article › peer-review

80 Scopus citations

Abstract

Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble fraction of rat lens. The estimated molecular weight of rat lens calpain II was 120 000, and the enzyme was composed of 80 000 and 28 000 MW subunits. Calpain II required 400 μm calcium, a reducing agent, and pH = 7·5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat α-, β_H-, and β_L-crystallins, insoluble proteins, and intrinsic membrane proteins. γ-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation.

Original language	English (US)
Pages (from-to)	227-238
Number of pages	12
Journal	Experimental Eye Research
Volume	42
Issue number	3
DOIs	https://doi.org/10.1016/0014-4835(86)90057-6
State	Published - Mar 1986

Keywords

calpain II
cataract
protease
protein degradation
rat lens

ASJC Scopus subject areas

Ophthalmology
Sensory Systems
Cellular and Molecular Neuroscience

Access to Document

10.1016/0014-4835(86)90057-6

Cite this

@article{b3f7ef6a5f3b4a10bf0852ff9683e5f3,

title = "Purification of calpain II from rat lens and determination of endogenous substrates",

abstract = "Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble fraction of rat lens. The estimated molecular weight of rat lens calpain II was 120 000, and the enzyme was composed of 80 000 and 28 000 MW subunits. Calpain II required 400 μm calcium, a reducing agent, and pH = 7·5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat α-, βH-, and βL-crystallins, insoluble proteins, and intrinsic membrane proteins. γ-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation.",

keywords = "calpain II, cataract, protease, protein degradation, rat lens",

author = "David, {Larry L.} and Shearer, {Thomas R.}",

year = "1986",

month = mar,

doi = "10.1016/0014-4835(86)90057-6",

language = "English (US)",

volume = "42",

pages = "227--238",

journal = "Experimental Eye Research",

issn = "0014-4835",

publisher = "Academic Press Inc.",

number = "3",

}

TY - JOUR

T1 - Purification of calpain II from rat lens and determination of endogenous substrates

AU - David, Larry L.

AU - Shearer, Thomas R.

PY - 1986/3

Y1 - 1986/3

N2 - Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble fraction of rat lens. The estimated molecular weight of rat lens calpain II was 120 000, and the enzyme was composed of 80 000 and 28 000 MW subunits. Calpain II required 400 μm calcium, a reducing agent, and pH = 7·5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat α-, βH-, and βL-crystallins, insoluble proteins, and intrinsic membrane proteins. γ-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation.

AB - Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble fraction of rat lens. The estimated molecular weight of rat lens calpain II was 120 000, and the enzyme was composed of 80 000 and 28 000 MW subunits. Calpain II required 400 μm calcium, a reducing agent, and pH = 7·5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat α-, βH-, and βL-crystallins, insoluble proteins, and intrinsic membrane proteins. γ-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation.

KW - calpain II

KW - cataract

KW - protease

KW - protein degradation

KW - rat lens

UR - http://www.scopus.com/inward/record.url?scp=0022541892&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022541892&partnerID=8YFLogxK

U2 - 10.1016/0014-4835(86)90057-6

DO - 10.1016/0014-4835(86)90057-6

M3 - Article

C2 - 3011481

AN - SCOPUS:0022541892

SN - 0014-4835

VL - 42

SP - 227

EP - 238

JO - Experimental Eye Research

JF - Experimental Eye Research

IS - 3

ER -

Purification of calpain II from rat lens and determination of endogenous substrates

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this