The optimal activity of a pseudozymogen form of recombinant matriptase under the mildly acidic pH and low ionic strength conditions.
Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e. conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-l-Lys-l-Thr-l-Lys-l-Gln-l-Leu-l-Arg-4-methyl-coumaryl-7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate pH approximately 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only approximately 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.
Inouye
Kuniyo
K
Laboratory of Enzyme Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto City 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Yasumoto
Makoto
M
Tsuzuki
Satoshi
S
Mochida
Seiya
S
Fushiki
Tohru
T
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D004765:Enteropeptidase; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009123:Muscle Hypotonia; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000590:Amino Acid Chloromethyl Ketones; D004792:Enzyme Precursors; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C086298:glutamyl-glycyl-arginyl-chloromethyl ketone; D012697:Serine Endopeptidases; C485595:matriptase; D004765:Enteropeptidase