JOHN BUTTERWORTH and DAVID A. PRI ESTM A N. Department of Genetic Pathology, Royar' Hospital for Sick. Children and University of Edinburgh, ...
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609th MEETING, LEEDS
Prolidases of human skin fibroblasts JOHN BUTTERWORTH and DAVID A. PRI ESTM A N Department of Genetic Pathology, Royar' Hospital for Sick Children and University of Edinburgh, Edinburgh EH9 ILF, Scotland. U .K .
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// Prolidase deficiency (McKusick 2641 3) is an autosomal I ' recessive disease of humans associated with chronic I 1 / t ulcerative dermatitis, mental retardation and iminodipepti/ I 1 / 200 duria (Scriver, 1977). In the disease, cultured skin a I I / E I fibroblasts have been shown (Sheffield let al., 1977; Arata et E I 1 l al., 1979; Myara et al., 1983) to have little or no prolidase 1 (EC 3.4.13.9) activity against glycyl-L-proline. However, we E 5 25have found (Butterworth & Priestman. 1984; Priestman & E a, -, Butterworth, 1984) that the activity against other substrates u ia was only reduced 30-80%, with the reduction being the Z largest for N-terminal polar amino acids. / 100 Control prolidase was activated by the addition of MnZ+ Y / with the substrate and even more so after preincubation wC / I with Mn?+. However, the abnormal enzyme required a ,/ I I / much lower level of Mn2+ when added with the substrate / I and was inactivated by preincubation with the usual level of Mn?+.The affinity of the abnormal enzyme for a number of substrates was reduced with, for example, the K , for leucylL-proline being increased from 1.3 to 1 0 . 4 m ~Heat . stability 0 (48°C) of normal prolidase activity was increased, but 0 30 60 abnormal prolidase activity decreased in the presence of Vol. (tnl) Mn2+.Inhibition of normal prolidase by p-hydroxymecuribenzoate (0.05mM) was largely prevented by preincubation Fig. 1. Cultured skinJibroblast prolidase separated by DEAEof the enzyme with Mn'+, whereas the abnormal enzyme cellulose chromatography was almost totally inactivated whether or not MnZ+was present. These results indicate that either a single form of Prolidase was assayed with glycyl-L-proline ( x --- x ) and prolidase is altered kinetically or that there are two forms of phenylalanyl-L-proline (0-0). prolidase, at least one of which is alte:red in the disease. To decide between these two alternatives, the separation of fibroblast prolidase into different forms was attempted by using ion-exchange chromatography. Fibroblasts were against glycyl-L-proline, but not the other substrates tested. cultured and cell extracts prepared as before (Butterworth, The abnormal prolidase second peak showed a reduced 1982; Butterworth & Priestman, 1982). DEAE-cellulose affinity for phenylalanyl-L-proline and decreased heat ion-exchange chromatography was caxried out as before stability (48"C), as compared with that of the control. Hence (Butterworth & Priestman, 1982), except the sample was there are two forms of prolidase in cultured skin fibroblasts, applied without prior dialysis and prolidase assayed with a both showing changes in the disease indicating them to be number of substrates (Butterworth & Priestman, 1984). Two inter-related. peaks of prolidase activity were obtained eluting at 135 and 2 2 0 m ~ - N a C (Fig. 1 1). Peak 1 was very active against glycylThe authors acknowledge the financial assistance of The L-proline compared with phenylalanyl-L-proline, whereas Scottish Home and Health Department. peak 2 was relatively less active against glycyl-L-proline. The activity against phenylalanyl-L-proline was split about Arata, J., Umemura, S . , Yamamoto, Y., Hagiyama, M. & Nohara, N. (1979) Arch. Dermatol. 115, 62-67 60% peak 1 and 40% peak 2. Both peaks were also active Butterworth, J. (1982) J . Inherited Metab. Dis. 5, 187-191 against leucyl-L-proline, alanyl-L-proline, valyl-L-proline J. & Priestman, D . (1982) Clin. Chim. Acra. 122, 51and methionyl-L-proline. A previous inability to detect the Butterworth, 60 second peak in fibroblasts (Butterworth & Priestman, 1984) Butterworth, J. & Priestman, D . (1984) J. Inherited Metab. Dis. 7 , was due to dialysis of the extract before chromatography, as 32-34 it was found that peak 2 activity was lost by dialysis. Endo, F., Matsuda, I., Ogata, A. & Tanaka, S. (1982) Pediatr. Res. Although a sulfhydryl reagent was not needed for peak 2 16, 227-231 activity, the inclusion of 0.1 mM-dithiothreitol during Myara, I., Charpentier, C., Wolfrom, C., Gautier, M., Lemonnier, A,, Larregue, M., Chamson, A. & Frey, J. (1983) J. Inherited dialysis prevented its loss. We found that human Metab. Dis. 6, 27-31 erythrocytes contained about 10% pea.k 2 activity against Priestman, D. A. & Butterworth, J . (1984) Clin. Cheni. Acra 142, phenylalanyl-L-proline, and the previous failure (Endo et 263-27 1 al., 1982) to demonstrate this peak 2 activity was probably Scriver, C. R. (1977) in The Metabolic Basis of Inherited Disease due to the low level measurable with glycyl-L-proline and (Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D . S., eds.), the preparative procedure used. The fibroblast prolidase pp. 355-357, McGraw-Hill, New York activity of two unrelated cases with prolidase-deficiency Sheffield, L. J., Schlesinger, P., Faull, K., Halpern, B. J., Schier, showed almost a total lack of peak 1 activity against all the G . M., Cotton, R. G . H., Hammond, J. & Danks, D . M. (1977)J. Pediatr. 91, 578-583 substrates, whereas peak 2 showed much reduced activity
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