Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin

Abstract

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported.

Cloning and Sequencing of the Gene Encoding the Spike Protein of the Coronavirus IBV

“…Following incubation for 90rain at 56 °C under nitrogen the glass was washed with water and methanol to remove non-covalently bound material. The glass-coupled peptide was then sequenced by automated solid-phase Edman degradation (Brett & Findlay, 1983).…”

Sequence variability in the retinal-attachment domain of mammalian rhodopsins

“…The V8-cleaved [3H]retinal-rhodopsin complex was purified by using concanavalin A-Sepharose as described previously (Van Breugel et al, 1977;Brett & Findlay, 1983 (Bownds & Wald, 1965;Akhtar et al, 1965). The solution became colourless in 15-20s, but reduction was allowed to continue for a period of 2min before the pH of the solution was decreased to below pH 3 with small quantities of 6M-HCI to hydrolyse the excess borohydride.…”

Cloning and sequence analysis of the human brain β‐adrenergic receptor

“…The hydropathy profile, based upon the analysis of Kyte and Doolittle [16], of the human brain ,& receptor is nearly identical to that of the hamster lung &receptor [6] and has features similar to those of the turkey &receptor [7], the pig brain muscarinic receptor [8] and the rhodopsins from human [17], cow [18], sheep [19,20], fruitfly [211 and bacteria [22] (not shown). The overall homology of these proteins is shown in table 1.…”

“…The higher homology seen between our gene and the [18], sheep [19,20], fruitfly [21], bacteria [22] hamster lung &receptor as compared to the turkey erythrocyte 'PI-like receptor could imply that we have cloned a human &-receptor gene. On the other hand, the degree of homology could merely reflect species variation and evolution of these proteins.…”