Immunological distinction between piscine and mammalian metallothioneins.

1. Separate antisera to metallothioneins (MT) from rainbow trout and horse were produced in mice and their reactivity with the respective immunogen was confirmed using an ELISA. 2. The ELISA, used in a competitive mode, revealed that the anti-horse MT serum did not cross-react with trout MT. Reciprocally, the anti-trout MT serum did not show any reactivity with horse MT. 3. The anti-rainbow trout MT serum was shown to cross-react totally with MTs from plaice, flounder, turbot, perch, salmon and pike, but exhibited no reactivity towards MTs from human, mouse, rat, worm or crab. Partial reactivity with the proteins isolated from oyster and mussel was demonstrated. 4. The anti-horse MT serum cross-reacted totally with MTs from human, rat and rabbit but no reactivity was demonstrable when MT from either plaice or worm was tested. 5. The behaviour of apo-, holo- and recombinant rainbow trout MTs, in which the metal content was different, indicated that reactivity with anti-MT antibodies was not dependent on the presence or nature of the metals bound in the protein. 6. The patterns of reactivities were analysed in relation to the known amino acid sequences of MT.

Purification and properties of cathepsin D from rat Yoshida ascites hepatoma AH-130.

Cathepsin D was affinity-purified on pepstatin-Sepharose from control rat liver, from Yoshida ascites hepatoma (AH-130) cells, and from the liver of AH-130 tumour-bearing rats. Apparent molecular mass and immunological reactivity, as determined by SDS-PAGE and immunoblotting, were identical for the three enzyme preparations. The active enzyme concentrations were determined by active-site titration. Catalytic parameters were measured for the three enzymes using two synthetic chromogenic peptides as substrates, and inhibition constants were determined for the proteinases with a number of naturally-occurring as well as synthetic inhibitors. All three enzymes were clearly distinguished from cathepsin E, since none of them was affected by the protein inhibitor from Ascaris lumbricoides. The cathepsin D isolated from AH-130 cells was indistinguishable in its kinetic properties from rat liver cathepsin D, except in its susceptibility to inhibition by isovaleryl-pepstatin. On isoelectrofocusing, the isoenzyme pattern of the tumour enzyme was shifted somewhat towards more basic pI values by comparison with rat liver cathepsin D. These findings are considered with respect to the possibility of an alteration in the S4 subsite of the enzyme active site cleft.