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Clinical Scfence(1979) 5'1,1099-1 10s Renin precursor synthesis and renin-binding proteins in the mouse K. POULSEN Institutefor Biochemistry, The Royal Dental College, Copenhagen, Denmark Summary 1. The precursor synthesis of renin, the storage form in the kidney and the submaxillary gland, and the molecular nature of the forms in plasma were studied in the mouse. 2. Renin is synthesized as a precursor (pre-prorenin) with a molecular weight of 50 000. 3. Renin is stored in the submaxillary gland and the kidneys as fully active renin with a molecular weight of 40 000. 4. The predominant form of renin in plasma is the active mol. wt. 40000 form. High-molecularweight forms of renin (800000 and 70000) are also present in plasma. 5. Pure 'Wabelled mol. wt. 40 000 renin binds, after a change in the tertiary structure, to the plasma protease inhibitors q-macroglobulin, intercetrypsin inhibitor and q-antithrombin. It binds also to lipoprotein and an unidentified plasma protein. No binding was seen to more than 50 other studied plasma proteins. 6. The high-molecular-weight forms of renin in plasma may be complexes of renin with plasma protease inhibitors and lipoprotein. Key words: lipoproteins, protease inhibitors, renin. Introduction High-molecular-weight forms of renin which are partly inactive are found in plasma of many species, including the .mouse (Nielsen, Malling & Poulsen, 1978). Such renin forms can either be precursors of renin or renin-protein complexes. In order to assess the nature of these renin forms, the Correspondence: Dr K. Poulsen, Institute for Biochemistry, The Royal Dental College, Jagtvej 160,2100 Copenhagen 0, Denmark. biosynthesis, the storage form and the binding of renin to other proteins were investigated. The mouse was chosen for these studies because of the availability of pure s u b m d a r y renin and its specific antibody, which were essential tools for elucidation of the molecular changes of renin from synthesis to elimination. Results and discussion Proteins are synthesized in the cytoplasma of the cell by the ribosomes, which use RNA as a messenger for the amino acid sequence. Ribosomes from one species and messenger RNA from another can interact and produce a protein with the correct structure. This is used in the so-called cellfree translation systems. The two most commonly used systems are the wheat-germ system (Rowe, Moen, Davidson, Byers, Bornstein & Palmiter, 1978) and the reticulo-lysate system (Housman, Jacobs-Lorena, Rajbhandary & Lodish, 1970). Both are characterized by having all the potentials for synthesizing proteins, and they translate messenger RNA added to them. Furthermore, an important feature is that they are derived from nonsecretory tissues, which means that they are not processing the proteins synthesized. If a messenger RNA from a different species is translated in these cell-free systems, they are left in the solution in the molecular form coded for. In order to study the molecular form in which renin is synthesized, the messenger RNA species from renin-rich submaxillary glands of the mouse were purified and translated in the two cell-free translation systems mentioned above. (Poulsen, Vuust, Lykkegaard, Nielsen & Lund, 1979b). The messenger RNA from the submaxillary gland were purified from other RNA species, but there is no means to purify specifically that particular messenger RNA which codes for renin. Renin will be 109s 110s K.Poulsen synthesized together with all other proteins synthesized in the submaxillary gland. However, the synthesized renin was purified by precipitation with a specific antibody against pure submaxillary renin. In order to visualize the synthesized renin [35S]methioninewas used in the protein synthesis. The molecular weight was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. In both assay systems renin was synthesized with a molecular weight of 50 000, which is 10 OOO greater than the fully enzymatic active mol. wt. 40000 renin (Poulsen et al., 1979b). Control experiments demonstrated that it was displaced from the antibody by pure submaxillary renin and that it was not precipitated by non-immune yglobulin. This mol. wt. 50 000 renin form represents most likely a pre-prorenin. It is unlikely that it is derived from an even larger precursor, since the two cellfree systems used gave the same molecular weight, and it is very unlikely that they contain the necessary processing enzymes. Other acid proteases, the group to which renin belongs, e.g. pepsin and chymosin (Foltman, Pedersen, Jacobsen, Kauffman & Wybrand, 1977) are also synthesized as a single-chain precursor polypeptide. Pepsinogen is stored in this precursor form, and secreted from the cell in that form. However, renin seems to be stored in both the submaxillary gland (Nielsen, Lykkegaard & Poulsen, 1979) and the kidney (K. Poulsen, unpublished work) of the mouse as fully enzymically active mol. wt. 40 OOO renin. Thus it is most likely that renin is secreted into the blood as fully active mol. wt. 40 OOO renin. This is in accordance with the findings that the majority of renin in mouse plasma is present as fully active renin. That the molecular weight is 40 000 is determined by gel chromatography, and that it is fully active is determined by measuring its specific enzymatic activity (Nielsen et al., 1978). However, in plasma of the mouse two distinct high-molecular-weight forms of renin are present (Nielsen et al., 1978). One has mol. wt. 800000 and the other 70 000. Such high-molecular-weight forms of renin are most likely complexes of renin with other molecules, since they are greater than the mol. wt. 50000 form in which renin is sykhesized. If pure submaxillary renin is mixed with mouse plasma it does not bind to other proteins. This facilitates its enzymatic activity in mouse plasma, but does not offer an explanation for the highmolecular-weight forms. In order to mimic complex-formation of renin with plasma proteins 1251-labelled pure submaxillary mol. wt. 40000 renin was mixed with mouse plasma after different kinds of treatment in order to provoke binding. Unfolding and subsep e n t refolding of renin by guanidine facilitated binding to plasma proteins. The binding proteins nere identified as the plasma protease inhibitors qmacroglobulin, inter-ct-trypsin inhibitor and qantithrombin. Binding to a,- and /],-Lipoproteins and to an unidentified plasma protein was also demonstrated. There was no binding to 56 other tested plasma proteins (Poulsen, Kr011, Nielsen, Jensenius & Malling, 1979a). The physiological relevance of the renin-protein complexes is still unknown. References FOLTMAN,B., PEDERSEN, V.B., JACOBSEN, H., KAUFFMAN, D. & WYBRAND, G. (1977) The complete amino acid sequence of prochymosin. Proceedings of the National Academy of Sciences U S A . , 74,2321-2324. HOUSMAN, D. JACOBS-LORENA, M., RAJBHANDARY, U.L. & LODISH,H.E. (1970) Initiation of haemoglobin synthesis by methionyl-tRNA. Nature (London), 227.9 13-918. S.& POULSEN, K. (1979) Renin NIELSEN,A.H., LYKKEGAARD, in the mouse submaxillary gland has a molecular weight of 40 000. Biochimica el Biophysica Acta, 516,305-3 13. NIELSEN,A.H., MALLMG,C. & POULSEN,K. (1978) Characteristics and conversion of high molecular weight forms of renin in plasma and their uncomplete activation by the current acid treatment. Biochimica et Biophysica Acta, 534, 246-257. POULSEN,K.. KRBLL, J., NIELSEN,A.H., JENSENIUS, J. & MALLING,C. (1979a) Renin binding proteins in plasma. Binding of renin to some of the plasma protease inhibitors, to lipoproteins, and to a non-trypsin-binding unidentified plasma protein. Biochimica et Biophysica Acta, 577, 1-10. POULSEN, K.. VUUST,J., LYKKEGAARD, S., NIELSEN,A.H. & LUNO,T. (1979b) Renin is synthesized as a 50000 dalton single-chain polypeptide in cell-free translation systems. FEES Letters, 98,135-138. ROWE, D.W., MOEN, R.C., DAVIDSON,J.M., BYERS, P.M., BORNSTEIN, P. & PALMITER,R.D. (1978) Correlation of procollagen mRNA levels in normal and transformed chick embryo fibroblasts with different rates of procollagen synthesis. Biochemistry. 17, 1581-1590.