Fleminger G, Yaron A
Biochim Biophys Acta. 1983 Mar 30;743(3):437-46. doi: 10.1016/0167-4838(83)90403-x.
Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.
含脯氨酸的多肽被证明可被两种氨肽酶依次降解。梭菌氨肽酶（EC 3.4.11 -）从多肽链上切割下任何N端氨基酸残基，包括脯氨酸，但不切割涉及脯氨酰氮的N端二级肽键。氨肽酶P（EC 3.4.11.9）专门切割此类二级键。通过将这两种酶共价偶联到多孔氨基玻璃珠上使其固定化。获得了具有不变的最适pH和热稳定性的高度稳定制剂。通过脑啡肽和P物质八肽的时间依赖性水解证明了梭菌氨肽酶在序列测定中的适用性。用含脯氨酸的（Pro - Gly - Pro）10、[Asn1，Val5]血管紧张素II、缓激肽、P物质和tuftsin证明了用两种固定化酶进行的顺序水解。通过细胞色素c对水解的抗性以及在固定化梭菌氨肽酶和氨肽酶P顺序降解过程中氨基酸的有序释放证明了不存在内肽酶活性。
