Hui Maria, Hui Koon-Sea
Peptide Research Laboratory, Neurochemistry Division, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, 10962, USA.
Neurochem Res. 2006 Jan;31(1):95-102. doi: 10.1007/s11064-005-9234-9.
Neuropeptides are formed from sedentary precursors to smaller, active peptides by processing enzymes cleaving at paired basic residues. The process generates peptide intermediates with additional Lys or Arg residues at their NH(2) and COOH termini; the N-terminal basic amino acids are later removed by specific aminopeptidases. We report here a novel lysine-specific aminopeptidase (KAP) of ubiquitous distribution. The enzyme was resolved from puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB), and neuron-specific aminopeptidase (NAP). It was purified by FPLC after (NH(4))(2)SO(4) precipitation. The purified KAP had a K(m) of 333 microM with a V(max) of 0.7 nmol Lys ssNA/min/mg protein. N-terminal basic amino acids, Lys in particular, were its favorable substrates. KAP was inhibited by chelating agents and by serine protease inhibitors. It was highly sensitive to aminopeptidase inhibitor bestatin, but insensitive to puromycin and amastatin, showing that KAP is distinct from PSA, NAP, and aminopeptidase A (APA). The 62,000-Da enzyme had a pH optimum at 7.5 and NaCl was its strongest activator. However, metals could not restore KAP's activity after it was dialyzed against EGTA. Our data indicated that rat KAP did not resemble any aminopeptidases as well as the microbial lysine aminopeptidases.
神经肽是由无活性的前体经加工酶在成对碱性残基处切割形成较小的活性肽。该过程产生在其NH(2)和COOH末端带有额外赖氨酸或精氨酸残基的肽中间体；N端碱性氨基酸随后被特定的氨肽酶去除。我们在此报告一种分布广泛的新型赖氨酸特异性氨肽酶（KAP）。该酶与嘌呤霉素敏感氨肽酶（PSA）、氨肽酶B（APB）和神经元特异性氨肽酶（NAP）不同。经硫酸铵沉淀后通过快速蛋白质液相色谱法（FPLC）进行纯化。纯化后的KAP对赖氨酸的Km为333微摩尔，Vmax为0.7纳摩尔赖氨酸底物类似物/分钟/毫克蛋白质。N端碱性氨基酸，尤其是赖氨酸，是其适宜的底物。KAP受到螯合剂和丝氨酸蛋白酶抑制剂的抑制。它对氨肽酶抑制剂苯丁抑制素高度敏感，但对嘌呤霉素和抑氨肽酶素不敏感，表明KAP与PSA、NAP和氨肽酶A（APA）不同。这种62,000道尔顿的酶在pH 7.5时活性最佳，氯化钠是其最强的激活剂。然而，在用乙二醇双四乙酸（EGTA）透析后，金属无法恢复KAP的活性。我们的数据表明，大鼠KAP与任何氨肽酶以及微生物赖氨酸氨肽酶均不同。
