The metalloenzymic nature of collagenase-like peptidase of the rat testis

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

Section: Influence Of Metal Ionssupporting

confidence: 93%

Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit

Tisljar¹,

Barrett²

1990

“…Inactivation of the enzyme by chelating agents has previously been reported by Lukac & Koren (1977), Morales & Woessner (1977) and Orlowski et al (1983). In some studies inactivation by EDTA has not been seen, and the effect of 1,10-phenanthroline has been reversible by dialysis without the addition of metal ions (Camargo et al, 1973;.…”

Section: Removal Of Essential Metal Ionmentioning

confidence: 76%

Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase

Barrett¹,

Brown²

1990

Pz-peptidase was purified from chicken liver as a protein of Mr 80000 and pl 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys, 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitrophenyl)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-I-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed K1 values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. pChloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pzpeptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree well with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A, consistent with the view that these three types of activity are all attributable to the single enzyme for which the niame thimet peptidase has been proposed.

“…An unusual property common to both Pz-peptidase and endopeptidase 24.15 is that of activation by dithiothreitol at low concentrations and inhibition at higher concentrations (Rajabi & Woessner, 1984;. Both activities are also inhibited by 1,10-phenanthroline and EDTA, and re-activated by metal ions after the latter (Lukac & Koren, 1977;Chu & Orlowski, 1983).…”

Section: Discussionmentioning

confidence: 99%

The activities of ‘Pz-peptidase’ and ‘endopeptidase 24.15’ are due to a single enzyme

Barrett¹,

Tisljar²

1989

It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.