Further studies on the sub-units of α-crystallin

Abstract

1. A new procedure is described for the purification of alpha-crystallin, including: preparative zone electrophoresis, density-gradient centrifugation and gel filtration. The total amino acid composition of highly purified samples prepared according to this procedure has been determined. 2. Evidence is presented for the presence of intermediates in the urea-induced splitting of alpha-crystallin into sub-units. A possible mechanism for this splitting is proposed. 3. The recombination of sub-units has been studied by polyacrylamide-gel electrophoresis and ultracentrifugal analysis. As judged from these criteria, only a partial recovery of starting material was obtained. 4. The origin of the minor bands in the electrophoretic pattern of alpha-crystallin on 7m-urea-polyacrylamide gel has been investigated. No evidence was found that their presence is due to carbamoylation or sulphide-disulphide interchange. They probably arise from isomerization. 5. The mean molecular weight of the sub-units was calculated to be 24000 (Archibald's method). Determination of the sedimentation-diffusion equilibrium revealed a value of 21000 at the meniscus. Assuming that all sub-units contain one cysteine residue/molecule, 23000 can be derived for the mean molecular weight.

Isolation of Non‐identical Polypeptide Chains of Α‐crystallin

“…The mean molecular weight of the subunit as calculated by approach to equilibrium methods is about 24,000 (Bont et al 1962, Wisse et al 1966. The mean molecular weight of the subunit as calculated by approach to equilibrium methods is about 24,000 (Bont et al 1962, Wisse et al 1966.…”

Proceedings of the Physiological Society, 12‐13 July 1968, Edinburgh Meeting: Demonstrations

“…A subunit structure occurs in the oc-crystallins of bovine lens (Wisse, Zweers, Jongkind, Bont & Bloemendal, 1966) and in the ,-crystallins (also called FISC or &-crystallins) of the chick (Clayton & Truman, 1967). It also appears probable that the y-crystallins (known also as /8-or 'long line' crystallins) of the chick are composed of subunits.…”

Structural Studies on Lens Proteins

“…Bovine ac-crystallin contains 1 cysteine residue/20 000 g. of protein (Thomann, 1959;Wisse et al 1966) and partial acid hydrolysis of the carboxymethylated protein gives Ser-CMOys (Waley, 1965a). This is a characteristic of bovine a-crystallin shared by human a-crystallin and by bovine and human albuminoid.…”

“…Bjork (1964) found that the early-peak protein aggregated (after the urea had been removed by dialysis) and he obtained S20.,, 13*2s, a value comparable with those obtained from the late-peak proteins. Moreover, the early-peak proteins represent the slowest bands seen on electrophoresis of a-crystallin in polyacrylamide gel in 7M-urea (Bjork, 1964;Wisse et al 1966); these bands are present when extensively purified a-crystallin is used, and Wisse et al (1966) concluded that the complexity of the electrophoretic pattern was not due to impurities in acrystallin. The amino acid composition ofthe earlypeak protein (Bjork, 1964;Spector, 1965) is consistent with this conclusion: proline and phenylalanine are relatively abundant, glycine and tyrosine are relatively scarce; these are features that distinguish a-crystallin from fi-crystallin and y-crystallin.…”

“…a-Crystallin is a large molecule, made up of 30-50 sub-units (Bloemendal, Bont, Jongkind & Wisse, 1962;Bjork, 1964;Bloemendal, Bont, Benedetti & Wisse, 1965;Spector & Katz, 1965;Wisse, Zweers, Jongkind, Bont & Bloemendal, 1966), and often obtained in several forms. Chromatography of bovine acrystallin, for example, gives two (Bjork, 1963) or four (Papaconstantinou, Resnik & Saito, 1962;Spector, 1965) peaks, and the nature of the differences between the fractions is not clear.…”