Purification of rabbit brain endooligopeptidases and preparation of antienzyme antibodies

Abstract

Endooligopeptidase A was purified approximately 3 000-fold and endooligopeptidase B approximately 1200-fold from the 25 000g supernatant fraction of rabbit brain homogenate. The purified enzymes were homogeneous on the basis of acrylamide gel electrophoresis under denaturing and nondenaturing conditions, isoelectric focusing, immunochemical criteria, and specific activities of the elution profile of gel filtration on Sephadex G-100. The only peptide bond cleaved by endooligopeptidase A in bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, is Phe5-Ser6, whereas endooligopeptidase B hydrolyzes the Pro7-Phe8 peptide bond of bradykinin and the Pro3-Gly4 bond of des-Phe8-Arg9-bradykinin. The specific activity of the homogeneous enzymes using bradykinin as substrate was 1087 nmol min-1 mg-1 for endooligopeptidase A and 292 nmol min-1 mg-1 for endooligopeptidase B. Gel filtration suggested molecular weights of 75 000 and 68 000 for endooligopeptidases A and B, respectively. Sodium dodecyl sulfate gel electrophoresis suggested that each endooligopeptidase consisted of a single polypeptide chain with molecular weights of 74 000 and 69 000 for the A and B enzymes, respectively. Purified endooligopeptidase A or B injected into goats produces monospecific antisera directed against each enzyme. The antibody prepared against each endooligopeptidase did not react with or inhibit the activity of the other enzyme.

Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase

“…The minimum substrate length of six amino acid residues in Bk and Dyn homologues required for hydrolysis by the recombinant rat testis TOP is similar to that previously observed with the purified rabbit brain endo-oligopeptidase A [2,11]. The core peptide sequence for the series of Dyn A " -) peptides is Gly-Phe-Leu-Arg- [1][2][3][4][5][6][7][8] …”

“…In the early 1970s two thiol-activated endopeptidases were isolated from the cytosol of rabbit brain [1,2] and subsequently purified to apparent homogeneity and characterized [3]. Unlike typical proteinases, these enzymes presented strict selectivity for oligopeptides and thus were generically named endo-oligopeptidases A and B [4].…”

“…An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin- (2)(3)(4)(5)(6)(7)(8) [qf-Dyn # -) ; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N- (2,4-dinitrophenyl) ethylenediamine], to Arg-Arg in qf-Dyn # -) Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gln at the C-terminal position, namely Abz-RPPGFSPFREDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQEDDnp are cleaved at the Pro-Phe bond.…”

Pathway for Degradation of Peptides Generated by Proteasomes

“…The biological function(s) of this enzyme have long been unclear. TOP was initially discovered by Camargo (44,45) and Orlowski (46) and their co- workers by its ability to degrade a variety of circulating peptides and has therefore been assumed to function primarily in the catabolism of neuropeptides. However, it is very unlikely that its primary role is in metabolism of extracellular peptides, since TOP is localized in most cells primarily in the cytosol and nucleus, and at most only about 5% of cellular TOP is found on the surface of neuroendocrine cells (47).…”

Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

“…These enzymes are easily distinguishable from other metallopeptidase s, such as neutral endopeptidase 24.11 or angiotensin I-converting enzyme, on the basis of their substrate and inhibitor specificity. The metalloendopep tidase 24.15 (EC 3.4.24.15; EP24.15) was initially described as endo-oligopeptida se A (Endo-A; formerly EC 3.4.22.19), a cytosolic thiol-dependent endopeptidase present in rabbit brain and responsible for the hydrolysis of the Phe 5 -Ser 6 bond of bradykinin (Camargo et al, 1973;Carvalho and Camargo, 1981). Subsequently, a cytosolic, thiol-activated metalloendopep tidase similar to Endo-A was isolated from rat brain (Orlowski et al, 1983).…”