A heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel-exclusion and anion-exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12.Calcium ions bind to the enzyme, pK,, 4.5, and activate it about sixfold when the substrate is leucine-4-nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25°C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two-stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions.Sensitive metalloendopeptidase assays are usually based on a two-stage enzymatic reaction in which the endopeptidase attack of a blocked peptide substrate is followed by the rapid action of an aminopeptidase, added externally, to generate a highly absorbing chromophore or a fluorophor [l, 21. The aminopeptidase most widely employed in such assays is aminopeptidase M [l, 3 -51, but this enzyme has a very high molecular mass and is difficult to purify from accompanying endopeptidases [5].In the course of our studies of the membrane-bound metalloendopeptidase (EC 3.4.24.1 1) we have searched for a reliable source of an efficient aminopeptidase for a two-stage assay of the enzyme. We found that the extracellular fluid of Streptomyces griseus cultures is a rich source of a very lowmolecular-mass and highly active aminopeptidase that can be readily purified due to its thermostability [6, 71. Further, the activity of the enzyme can be modulated by the binding of calcium ions, providing an excellent model for the study of proteolytic enzyme activation by calcium ions.

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass Eur. J. Biochem. 183, 471 -4771. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1 ,lo-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes.Streptomyces griseus aminopeptidase (S. griseus AP) is a heat-stable and a low-molecular-mass extracellular bacterial enzyme, distinct from the high-molecular-mass cytosolic (e. g. leucine aminopeptidase) or membrane-bound (e. g. aminopeptidase N) mammalian aminopeptidases [l -51. The complete amino acid sequence and three-dimensional structure of S. griseus AP are still not known. The enzyme is a zinc metalloenzyme inhibitable by 1 ,lo-phenanthroline, EDTA or EGTA and it can bind CaZ+ with a concomitant increase in its activity [4]. S. griseus AP rapidly hydrolyzes N-terminal hydrophobic amino acids like Leu. Due to the unique physicochemical properties of S. griseus AP and its high enzymic reactivity, it has been used as an effective additive in twostage assays of yet other peptidases, neutral endopeptidase and thermolysin [6, 71. In this communication, we investigated the specificity of S. griseus AP and the modulation of its activity by Ca2+ binding and by transition-metal-ion substitution. The spectrum of activities observed with the different substrates and the way by which these activities are influenced by metal ions

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-~nitroanilide supplemented with Streptomyces griseus aminopeptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5,25"C) with a catalytic efficiency (k,, = 1.2 x 107 s-r, K,,, = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (& = 1.2 x 1P s-l) but the overall efficiency is diminished by a higher K,,, (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of BuciNus cereus have been assayed sensitively for their neutral endopeptidase and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.Neutral endopeptidase; Neutral proteinase; Enkephalinase; Thermolysin; Common acute lymphoblastic leukemia antigen

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