PMID- 10067969
OWN - NLM
STAT- MEDLINE
DCOM- 19990310
LR  - 20131121
IS  - 0146-0404 (Print)
IS  - 0146-0404 (Linking)
VI  - 40
IP  - 3
DP  - 1999 Mar
TI  - Modifications to rat lens major intrinsic protein in selenite-induced cataract.
PG  - 657-67
AB  - PURPOSE: To identify modifications to rat lens major intrinsic protein (MIP)
      isolated from selenite-induced cataract and to determine whether m-calpain (EC
      3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS:
      Cataracts were induced in rats by a single injection of sodium selenite. Control 
      and cataract lenses were harvested on day 16 and dissected into cortical and
      nuclear regions. Membranes were washed with urea buffer followed by NaOH. The
      protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide
      (CNBr). Cleavage products were fractionated by high-performance liquid
      chromatography (HPLC), and peptides were characterized by mass spectrometry and
      tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation
      with purified enzyme, and peptides released from the membrane were analyzed by
      Edman sequencing. RESULTS: The intact C terminus, observed in the control nuclear
      and cataractous cortical membranes, was not observed in the cataractous nuclear
      membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C
      terminus of MIP in control and cataract nuclear regions. The major site of
      cleavage was between residues 238 and 239, corresponding to the major site of in 
      vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacrylamide gel
      electrophoresis and mass spectrometric analysis indicated that in vivo
      proteolysis during cataract formation also included sites closer to the C
      terminus not produced by m-calpain in vitro. Evidence for heterogeneous
      N-terminal cleavage was also observed at low levels with no differences between
      control and cataractous lenses. The major site of phosphorylation was determined 
      to be at serine 235. CONCLUSIONS: Specific sites of MIP N- and C-terminal
      cleavage in selenite-induced cataractous lenses were identified. The
      heterogeneous cleavage pattern observed suggests that m-calpain is not the sole
      enzyme involved in MIP C-terminal processing in rat lens nuclei.
FAU - Schey, K L
AU  - Schey KL
AD  - Department of Cell and Molecular Pharmacology and Experimental Therapeutics,
      Medical University of South Carolina, Charleston 29425, USA.
FAU - Fowler, J G
AU  - Fowler JG
FAU - Shearer, T R
AU  - Shearer TR
FAU - David, L
AU  - David L
LA  - eng
GR  - EY03600/EY/NEI NIH HHS/United States
GR  - EY10722/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Invest Ophthalmol Vis Sci
JT  - Investigative ophthalmology & visual science
JID - 7703701
RN  - 0 (Aquaporins)
RN  - 0 (Eye Proteins)
RN  - 0 (Membrane Glycoproteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Phosphoproteins)
RN  - 0 (aquaporin 0)
RN  - EC 3.4.22.- (Calpain)
RN  - HIW548RQ3W (Sodium Selenite)
SB  - IM
MH  - Animals
MH  - Aquaporins
MH  - Calpain/physiology
MH  - Cataract/chemically induced/*metabolism
MH  - Chromatography, High Pressure Liquid
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Eye Proteins/analysis/isolation & purification/*metabolism
MH  - Gas Chromatography-Mass Spectrometry
MH  - Lens, Crystalline/chemistry/*metabolism
MH  - Membrane Glycoproteins/analysis/isolation & purification/*metabolism
MH  - Peptide Fragments/analysis
MH  - Phosphoproteins/analysis/isolation & purification/*metabolism
MH  - Phosphorylation
MH  - Rats
MH  - Sodium Selenite
EDAT- 1999/03/06 00:00
MHDA- 1999/03/06 00:01
CRDT- 1999/03/06 00:00
PHST- 1999/03/06 00:00 [pubmed]
PHST- 1999/03/06 00:01 [medline]
PHST- 1999/03/06 00:00 [entrez]
PST - ppublish
SO  - Invest Ophthalmol Vis Sci. 1999 Mar;40(3):657-67.