Streptomyces griseus aminopeptidase is a calcium‐activated zinc metalloprotein

Abstract

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.

A Secreted Aminopeptidase of Pseudomonas aeruginosa

“…To examine whether the aminopeptidase is thermostable, the concentrated culture filtrates from strains FRD2, FRD740, and FRD2128 (wild type, ⌬lasB and ⌬lasA, respectively) were each incubated at 70°C for 1-3 h, conditions that have been used in the past to demonstrate the heat stability of enzymes such as SgAP and VpAP (25,26). Heating for up to 3 h had virtually no effect on the aminopeptidase activity, indicating that PaAP is resistant to heat.…”

“…VpAP and SgAP are also distinguished by their heat (70°C) stability (25)(26)(27). The substrate specificity of VpAP and SgAP is similar, both cleaving preferentially hydrophobic amino acids especially leucine occupying the N-terminal position of proteins or peptides (25,28,29).…”

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

“…Reactions were carried out at 30°C in 50 mM Tris-HCl/ 100 mM NaC1/1 mM CAC12/0.25 mM Hepes/0.005% Triton X-100, pH 7.5, containing 6.7 ,ug/ml Streptomyces griseus aminopeptidase I [19] when Leu is the C-terminal amino acid of the substrates (compounds 2, 4, 6, 8) or 26.8 pg/ml of the same aminopeptidase when Phe is the C-terminal amino acid (compounds 1, 3, 5, 7). Reactions were performed in 200 pl volumes in 96-well polystyrene microplates (Greiner, Frickenhausen, Germany), using a thermoregulated Thermomax Microplate Reader (Molecular Devices Corp., Menlo Park, CA).…”

Purification and Properties of an Extracellular Aminopeptidase from Streptomyces Lividans 1326

“…Two aminopeptidases (API and APII) purified from S. griseus were originally shown to require Ca2+ for their enzymic activity (Vosbeck et al, 1973). More recently, however, these LAPs were proved to be Zn2+-containing enzymes that are activated by calcium (Spungin & Blumberg, 1989). Our data suggest that the M, 34000 LAP from S. lividans is a zinc metalloproteinase which is stabilized, but not necessarily activated, by calcium.…”

“…The large difference in calculated M , from SDS-PAGE and Superose-6 may result from an incomplete protein denaturation or binding of SDS, or an atypical retardation of the enzyme on the gel-filtration support column (Superose-6). Interestingly, the two aminopeptidases from S. griseus displayed nearly identical anomalous elution profiles from Superose-12 gel filtration as compared to their M , values determined from SDS-PAGE, as did S. Zividans LAP (Spungin & Blumberg, 1989).…”