Hans C. Freeman. Department of Biochemistry, University of Sydney, NSW. 2006, Australia. * Current address: Department of Biochemistry, University.
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Biochemical Society Transactions (1999) 27
147 Structural Studies of E.coli AminopeptidaseP. Charles S . Bond*, Matthew C.J.Wilce, J.Mitchell Guss & Hans C. Freeman Department of Biochemistry, University of Sydney, NSW 2006, Australia. * Current address: Department of Biochemistry, University of Dundee. Aminopeptidase P (AMPP, EC 3.4.11.9) is a prolinespecific aminopeptidase which cleaves peptides of the type Xaa-'Pro-Yaa, where * marks the scissile bond. The structure has been solved by X-ray crystallographic methods for the native enzyme (2.0 A resolution), a dipeptide-inhibited form (2.3 A) and a low pH inactive form (2.7 A). The protein is formed of two domains. The larger Cterminal domain contains the dinuclear manganese centre which is the site of peptidase activity (and is homologous to the dinuclear cobalt centre of methionine aminopeptidase). The overall structure of both domains is homologous to that of creatinase, which is not a metalloenzyme. AMPP forms a tetramer (or rather, a dimer of dimers) which has the effect of extending the active site cavity to include residues from a total of three molecules. This may have some influence on substrate selectivity. As bradykinin, a vasoactive peptide, is degraded by both bacterial and mammalian AMPPs (it contains the sequence Arg-Pro-Pro), E.coZi AMPP is of interest as a model for indentifying and examining modes of inhibition of the human enzyme and, ultimately, aiding the discovery of possible treatments for hypertension.
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INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN (IRBP): PHOTOSENSITISED LIGHTINDUCED DAMAGE AND BINDING PROPERTIES. Irina B. Fedorovich, Carolvn A. Converse* and Mikhail A. Ostrovsky. Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin Str. 4, I 17334, Moscow and *Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, G4 ONR. IRBP is a 140 kDa protein located in the interphotoreceptor matrix between the photoreceptor outer segments and the pigment epithelium. It is responsible for transport of retinoids to and from the photoreceptor cells. As with many proteins, it is susceptible to damage by visible light. We wished to investigate whether the fact that retinoids are bound to this protein make it more susceptible to this photooxidation, and to determine the effect the light damage has upon IRBPs subsequent retinoid binding properties. IRBP prepared from bovine retinas was purified by Concanavalin A-Sepharose and DEAE-Sephacel chromatography. IRBP complexed to all-trans retinal was irradiated with blue light (ca. 365 nm) at 0.012W/cm2. Bound retinoids were then removed by treatment with charcoal-coated dextran, and the extent of light damage measured as loss of tryptophan fluorescence and titratable thiol groups. Binding properties were quantified by measuring enhancement of alltrans retinol fluorescence in the presence of irradiated and control IRBP. Retinal complexation to IRBPwas found to increase light damage markedly; about 33% of the retinoid-binding sites were lost, but the relativebinding affinity of the remaining sites was not substantially altered. This light damage occurred within the range of physiological levels of illumination, suggesting that this protein may be particularly susceptible, and its damage could lead to problems with regeneration of visual pigments.
