甲硫氨酰氨肽酶

甲硫氨酰氨肽酶在蛋白质加工过程中负责切除新生肽链N端的起始甲硫氨酸,是正常生理功能所必需的。烟曲霉素类血管生成抑制在体内的分子靶点是甲硫氨酰氨肽酶－2,根据甲硫氨酰氨肽酶的结构以及催化和抑制机制,可以设计和合成新型的血管生成抑制剂;以甲硫氨酰氨肽酶为分子靶点,通过最新发展的活性检测方法建立高通量药物筛选模型,可以寻找甲硫氨酰氨肽酶的小分子抑制剂用于抗肿瘤新药的开发。     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Aminopeptidase C of Aspergillus niger Is a Novel Phenylalanine Aminopeptidase

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either pNA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.     

Degradation of Dynorphin-Related Peptides by the Puromycin-Sensitive Aminopeptidase and Aminopeptidase M

Abstract: The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. K _i determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., K _i for dynorphin A-17 = 23–30 n M with the K _i increasing to 25–50 µ M for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, k _cat remains relatively constant; however, as the peptide size increases beyond a decapeptide, k _cat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that k _cat was greatest for the pentapeptide. Thus, if one considers k _cat/ K _m as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.     

Aminopeptidase from Sphingomonas capsulata

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min(-1) for glutamate. At pH 7.5 (pH optimum) and 25 degrees C, the kinetic parameters for alanine para-nitroanilide were found to be k(cat) = 7600 min(-1) and K(m) = 14 mm. For alanine beta-naphthylamide, they were k(cat) = 860 min(-1) and K(m) = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.     

Aminopeptidase Profiles of Various Bacteria

The aminopeptidase specificity of 24 strains of bacteria was determined fluorometrically by use of a series of alpha-amino acid beta-naphthylamides as substrates. Provided that strict control over medium and growth time was adhered to, a reproducible profile of aminopeptidase activity was obtained which could be used for the identification of bacteria.     

Aminopeptidase A is angiotensinase A

Summary Quantitative histochemical measurements of aminopeptidase A (APA; E.C.3.4.11.7) were done kinetically in the kidney glomeruli of rat and mouse with an instrumental setup consisting of a microdensitometer and a computer-supported morphometric system. The histochemical demonstration of APA was carried out using the simultaneous azo coupling technique (purest-grade Fast Blue B as coupling agent and -l-glutamic acid-4-methoxy-2-naphthylamide as substrate). The methodological studies show that APA activity is calcium-ion-dependent and increases linearly with the thickness of the tissue section (3–12 m) and that the time-course of APA activity as determined by linear regression is linear only for the first 1 to 2 min of the reaction. — Kinetic measurements indicate a 40% decrease in APA activities when -l-glutamic acid-4-methoxy-2-naphthylamide (-l-Glu-MNA) is replaced by -l-aspartic acid-4-methoxy-2-naphthylamide. When -l-Glu-MNA is replaced with l-alanine-4-methoxy-2-naphthylamide, which is a substrate of aminopeptidase M (APM) only very low reaction rates are measurable (about 1.4% of those with -l-Glu-MNA). 100 and 130 mM NaCl in the incubation medium increase APA activities by approximately 16%–17%. — To clarify the functional importance of APA in the kidney, their activities were measured under the influence of angiotensins. The glomerulus was selected as the measuring site, for besides APA it contains no APM or other peptidases that could degrade angiotensins (the glomerular dipeptidyl peptidase IV is not inhibited by angiotensin II). Using the Lineweaver-Burk plot, we determined a K _m of 0.16 mM for the APA in rat glomeruli and 0.14 mM in mouse glomeruli. The V _max in mouse glomeruli is 1.6 times higher than in rat glomeruli. Ang iotensin I, II and III competitively inhibit APA in the rat and mouse glomeruli. — With quantitative histochemical techniques it was possible to show that APA is equivalent to angiotensinase A (splitting off the N-terminal aspartic acid from angiotensin I and II).Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

An Aminopeptidase of Aspergillus sojae

An aminopeptidase was purified from Aspergillus sojae X–816. The molecular weight of the enzyme was estimated to be 220,000. The isoelectric point was at pH 5.3. The optimum pH for l-leucylglycylglycine was 7.5. The enzyme was stable up to 37°C against temperature treatment for 15 min. Some chelating agents inhibited the enzyme activity. The Km value for l-leucylglycylglycine at pH 7.5 and 37°C was 45 mm. The Km value for l-leucyl-β-naphthylamide at pH 7.0 and 37°C was 2.2 mm.     

Purification and Characterization of an Aminopeptidase, LPAase 2, from Euonymus Leaves

An aminopeptidase, LPAase 2, from the leaves of Euonymus alatusf. ciliato-dentatus was purified about 240-fold by a combinationof DEAE-cellulose and Sephadex G-100 column chromatographies.The molecular weight of LPAase 2 was estimated to be about 62,000,and the optimum pH for the hydrolytic activity against leucinep-nitroanilide(LPA) was 7.6. LPAase 2 hydrolyzed LPA, leucine-rß-naphthylamide(leucine-NA), phenylalanine-NA and tyrosine-NA. It was inhibitedstrongly by p-chloromercuribenzoate (PCMB), iodoacetic acidand heavy metal ions, but was not affected by thiol compoundsand metal-chelating reagents. Therefore, a sulfhydryl groupcould be involved in the active site of LPAase 2. None of themetal ions tested promoted LPAase 2 activity. The propertiesof LPAase 2 were compared with those of aminopeptidases reportedfor other plants. (Received November 24, 1983; Accepted April 16, 1984)     

Aminopeptidase activity in adult Schistosoma mansoni

An aminopeptidase activity capable of hydrolysing leucine 4-nitroanilide and alanine 4-nitroanilide at pH 7.0 was detected in saponin-CaCl2 extracts and homogenates of adult Schistosoma mansoni. The extracts were also capable of acting on synthetic dipeptides at the same pH, preferentially hydrolysing peptide bonds following leucine, alanine, or proline N-terminal residues. Imide bonds were not hydrolysed. The hydrolysis of leucine 4-nitroanilide was apparently stimulated by thiols, strongly inhibited by 1 mM 4-chloromercuric benzene sulfonic acid, and partially inhibited by 1 mM 1,10-phenanthroline.