Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor

Y. Nakamura, C. Fukiage, Hong Ma, M. Shih, M. Azuma, Thomas (Tom) Shearer

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82- induced proteolysis in rodent lenses may occur even in the presence of calpastatin.

Original languageEnglish (US)
Pages (from-to)155-162
Number of pages8
JournalExperimental Eye Research
Volume69
Issue number2
DOIs
StatePublished - Aug 1999

Fingerprint

Lenses
Calpain
Vimentin
Cysteine Proteases
Caseins
Immunoblotting
Isoenzymes
Proteolysis
calpastatin
calpain Lp82
Rodentia
Calcium
m-calpain
Proteins
calpain inhibitors
N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal

Keywords

  • Calpastatin
  • Casein zymography
  • Immunoblot
  • Lp82
  • M-calpain
  • Vimentin

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor. / Nakamura, Y.; Fukiage, C.; Ma, Hong; Shih, M.; Azuma, M.; Shearer, Thomas (Tom).

In: Experimental Eye Research, Vol. 69, No. 2, 08.1999, p. 155-162.

Research output: Contribution to journalArticle

Nakamura, Y. ; Fukiage, C. ; Ma, Hong ; Shih, M. ; Azuma, M. ; Shearer, Thomas (Tom). / Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor. In: Experimental Eye Research. 1999 ; Vol. 69, No. 2. pp. 155-162.
@article{0a226034b73f4c1f8b74af997c611872,
title = "Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor",
abstract = "The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82- induced proteolysis in rodent lenses may occur even in the presence of calpastatin.",
keywords = "Calpastatin, Casein zymography, Immunoblot, Lp82, M-calpain, Vimentin",
author = "Y. Nakamura and C. Fukiage and Hong Ma and M. Shih and M. Azuma and Shearer, {Thomas (Tom)}",
year = "1999",
month = "8",
doi = "10.1006/exer.1998.0686",
language = "English (US)",
volume = "69",
pages = "155--162",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor

AU - Nakamura, Y.

AU - Fukiage, C.

AU - Ma, Hong

AU - Shih, M.

AU - Azuma, M.

AU - Shearer, Thomas (Tom)

PY - 1999/8

Y1 - 1999/8

N2 - The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82- induced proteolysis in rodent lenses may occur even in the presence of calpastatin.

AB - The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82- induced proteolysis in rodent lenses may occur even in the presence of calpastatin.

KW - Calpastatin

KW - Casein zymography

KW - Immunoblot

KW - Lp82

KW - M-calpain

KW - Vimentin

UR - http://www.scopus.com/inward/record.url?scp=0032787439&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032787439&partnerID=8YFLogxK

U2 - 10.1006/exer.1998.0686

DO - 10.1006/exer.1998.0686

M3 - Article

C2 - 10433852

AN - SCOPUS:0032787439

VL - 69

SP - 155

EP - 162

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

IS - 2

ER -