Giardia lamblia: characterization of proteinase activity in trophozoites

Exp Parasitol. 1989 Feb;68(2):168-75. doi: 10.1016/0014-4894(89)90094-5.

Abstract

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arginine / analogs & derivatives
  • Arginine / metabolism
  • Benzoylarginine-2-Naphthylamide / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / metabolism*
  • Giardia / enzymology*
  • Hemoglobins / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Substrate Specificity

Substances

  • Hemoglobins
  • Benzoylarginine-2-Naphthylamide
  • Arginine
  • N(alpha)-benzoylarginineamide
  • Endopeptidases