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Comparative Study
. 1977 Feb 17;265(5595):658-9.
doi: 10.1038/265658a0.

Comparison of bacterial and animal rhodopsins by hydrogen exchange studies

J J Englander, S W Englander
Comparative Study

Comparison of bacterial and animal rhodopsins by hydrogen exchange studies

J J Englander et al. Nature. .
No abstract available

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Figures

Fig. 1
Fig. 1
Hydrogen–tritium exchange-out data for purple membrane suspensions at pH 5.0 (○) and pH 4.7 (●). The bacteria were grown and the membranes were isolated according to Osterhelt and Stoeckenius. After exchange-in at pH 10.5 (2mM alanine buffer) for 5 d at 4 °C, the purple membrane suspension was adjusted to the appropriate pH and passed through a medium Sephadex-G25 column (at low hydrostatic pressure; 0 °C; pH 5.0, 10 mM cacodylate or pH 4.7, 5 mM citrate) to remove free tritiated water and initiate exchange-out. HX methods were detailed elsewhere. Two to three drop fractions collected from the final columns were diluted to 0.5 ml with 0.1 M phosphate buffer at pH 7.4, and maintained at 0°C in order to measure absorbance. (Absorbance measurements decrease slowly with time at room temperature and are slightly dependent upon pH and salt concentration.) Protein concentration in the samples was then computed using a molar extinction coefficient of 7.2 × 104 at 570 nm, determined using the retinaloxime method of Wald and Brown and the thiobarbituric acid method of Futterman. (Total range on 4 preparations was 7.0–7.5 × 104.) Bound tritium in the samples was assayed by liquid scintillation counting. These data were used to compute the number of hydrogens per molecule still unexchanged as a function of exchange-out time.
Fig. 2
Fig. 2
Limited exchange-in/exchange-out data at pH 5.0 and 0 °C. Purple membrane suspensions were labelled at 0 °C for only 5 min at pH 5 (10 mM cacodylate) and then passed quickly through a Sephadex column to initiate exchange-out. A semilog plot of the resulting data is given in a and the isolated free peptide phase, obtained by subtracting the slow phase from the early time data, is in b.
Fig. 3
Fig. 3
Limited exchange-out data at pH 4.7 and 0°C. Exchange-in was for 10 min at pH 4.7 (5 mM citrate) and subsequent treatment was as for Fig. 2.
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References

    1. Downer NW, Englander SW. Nature. 1975;254:625–627. - PubMed
    1. Henderson R, Unwin PNT. Nature. 1975;257:28–32. - PubMed
    1. Yee RY, Englander SW, von Hippel PH. J molec Biol. 1974;83:1–16. - PubMed
    1. Bridgen J, Walker I. Biochemistry. 1976;15:792–798. - PubMed
    1. Osterhelt D, Stoeckenius W. Nature new Biol. 1971;233:149–152. - PubMed

Publication types