January 1994
Volume 35, Issue 1
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Articles  |   January 1994
Identification of origin of two polypeptides of 4 and 5 kD isolated from human lenses.
Author Affiliations
  • O P Srivastava
    Missouri Lions Eye Research Foundation, Columbia.
  • K Srivastava
    Missouri Lions Eye Research Foundation, Columbia.
  • C Silney
    Missouri Lions Eye Research Foundation, Columbia.
Investigative Ophthalmology & Visual Science January 1994, Vol.35, 207-214. doi:
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      O P Srivastava, K Srivastava, C Silney; Identification of origin of two polypeptides of 4 and 5 kD isolated from human lenses.. Invest. Ophthalmol. Vis. Sci. 1994;35(1):207-214.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To purify crystallin fragments (degraded polypeptides molecular weight < 18 kD) and identify their parent crystallins. METHODS: The purification of polypeptides with apparent molecular weights of 4 and 5 kD was carried out using three sequential steps: Sephadex G-50 chromatography under denaturing conditions, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and high-performance liquid chromatography using a C-18 column. The parent crystallins of the two polypeptides were identified by the Western blotting method using polyclonal antibodies raised against individual 4 and 5 kD polypeptides and by comparing N-terminal amino acid sequences of the polypeptides with crystallins. RESULTS: Two polypeptides of 4 and 5 kD were purified by the three sequential steps as described from water-soluble proteins of lenses from 60-80-year-old donors. Both purified polypeptides showed a single major peak during high-performance liquid chromatography on a C-18 column and also a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Western blot analyses showed maximum immunoreactivity of the anti-4 kD polypeptide antibody to a 22 kD species of beta-crystallin, whereas the anti-5 kD polypeptide antibody showed maximum reactivity to only the alpha B crystallin. These results were further confirmed during comparison of the N-terminal amino acid sequences of the two polypeptides with crystallins. Such comparison showed that the 4 kD polypeptide originated from beta A 3/A1 crystallin after cleavage at His187-His188 bond. Further, the 5 kD polypeptide was a fragment of alpha B crystallin that originated after cleavage at Val145-Asn146 bond. CONCLUSION: These results showed that specific bonds of beta A3/A1 and alpha B crystallins are posttranslationally cleaved in vivo to produce 4 kD and 5 kD polypeptides, respectively.

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