| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
In the present study, several properties of prolidases separated using high performance liquid chromatography from control cultured human skin fibroblasts and prolidase deficient fibroblasts were investigated, and analyses of iminodipeptides in the urine of patients with prolidase deficiency have been demonstrated using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system(LC/API-MS) The results were as follows
1. Crude enzyme solutions of prolidase were extracted from cultured human skin fibroblasts derived from control and prolidase deficient sisters. Two forms of prolidases (prolidase I and II)were partially purified by high performance liquid chromatography equipped with an ion exchange column. On gel filtration, the relative molecular weights of prolidase I and II were estimated to be MW=105,000 and 151,000, respectively. The substrate specificity of partially purified prolidase I and II in control fibroblasts was estimated against Gly-Pro
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, Ala-Pro, Met-Pro. Each form of prolidase differed in its substrate specificity. In prolidase deficient sisters, the elder with typical clinical manifestations and the younger with only slight clinical manifestations, the activity of prolidase I was absent. However, the activity of prolidase II was sufficiently present in both sisters. The substrate specificity of prolidase II in the patients was similar to that of control. No difference in substrate specificity was found between these two patients.
2. Analyses of standard iminodipeptides and iminodipeptides in the urine of patients with prolidase deficiency have been demonstrated using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system.The separation was carried out on a reversed phase column using 0.1% aqueous trifluoroacetic acid-methanol (70:30 or 80:20). Very intense quasi-molecular ions ([M+H]^+) of various standard iminodipeptides were observed by this method. The quasi-molecular ions [M+H]^+ of various iminodipeptides were also observed in the urine samples of patients with prolidase deficiency,and Gly-Pro,Ala-Pro,Val-Pro,Leu-Pro,Ile-Pro,Ser-Pro,Thr-Pro,Glu-Pro,Asp-Pro His-Pro,Lys-Pro,Pro-Pro and Tyr-Pro as iminodipeptides containing proline with C-terminal residue and Glu-Hyp,Pro-Hyp,Ile-Hyp and Gly-Hyp as iminodipeptides containing hydroxyproline with C-terminal residue were identified in the urine of patients with prolidase deficiency.
3. It has been reported that patients with prolidase deficiency excrete various iminodipeptides containing a carboxyl-terminal proline (hydroxyproline) in the urine. However, the methods used, namely, the amino acid or isotachophoretic analysis through hydrolysis or enzymatic digestion did no more than indirectly detect the iminodipeptides. In this study the liquid chromatography / atmospheric pressure ionization - mass spectrometry (LC/API-MS) which directly analyzed iminodipeptides by scanning the protonated molecular ions ([M+H]^+) could distinguished X-Pro from Pro-X on mass chromatograms and mass spectra. And this apparatus successfully quantified urinary iminodipeptides of patients with prolidase deficiency. A quantitative investigation revealed that a patient with severe clinical symptoms who was one of siblings with prolidase deficiency, excreted more iminodipeptides than the other who did not have serious symptoms. LC/API-MS also disclosed iminodipeptides (Gly-Hyp and Pro-Hyp) not only in the urine of patients but also in their mother and in normal volunteers. Patients excreted much more Pro-Hyp than normal volunteers, whereas no quantitative differences were between the mother and controls. Very low prolidase activity against X-Pro in patients causes their excretion of a large amount of X-Pro. Assay of prolidase activities in erythrocytes indicated that in patients, the activity against X-Hyp was extremely low and even in the mother and normal volunteers. it was remarkably low in comparison to the activity against X-Pro. Less
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