Functional and structural studies of alpha-crystallin from galactosemic rat lenses.
Chaperone-like activity and structural changes of lens alpha-crystallin from rats fed with galactose at various time intervals have been studied using high-performance liquid chromatograph (HPLC), circular dichroism (CD), and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence emission. It was found that chaperone-like activity of alpha-crystallin from galactose-fed rats toward dithiothreitol (DTT)-induced insulin B aggregation started to decrease after 3 weeks and decreased significantly after 5 weeks. Consistent results were observed in lens morphology, and lens opacity slightly developed after 3 weeks and became obvious after 5 weeks. HPLC analysis for chaperone function showed that the formation of high molecular weight aggregates (HMWA) of alpha-/gamma-crystallins decreases with the increase of galactose-feeding time, revealing that chaperone-like activity is concomitant with the formation of HMWA. Circular dichroism results showed the reduction of beta-sheet structure and loss of microenvironment of aromatic-type amino acids for opaque lenses, indicating alpha-crystallin's secondary and tertiary structure changed with the development of the lens opacity. ANS binding site estimated by Klotz equation showed it is 1.5 times higher at room temperature and is 2.4 times higher at 58 degrees C for age-matched normal alpha-crystallin than for 5-week galactose-fed lens alpha-crystallin, indicating opaque lens alpha-crystallin loses the ability to assemble into an appropriately placed hydrophobic regions. The overall results accordingly indicated that galactose-induced cataractous alpha-crystallin has disordered structure, leading to the loss of its chaperone-like activity.
Huang
F Y
FY
Department of Chemistry, National Cheng Kung University, Tainan, 70101, Taiwan. fhuang@mail.ncku.edu.tw
Ho
Y
Y
Shaw
T S
TS
Chuang
S A
SA
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D005690:Galactose / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D005693:Galactosemias / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology*; D007328:Insulin / Q000378:metabolism; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D007328:Insulin; D018832:Molecular Chaperones; C027132:1-anilino-8-naphthalenesulfonate; D004229:Dithiothreitol; D005690:Galactose