Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitochondrial proton-translocating nicotinamide nucleotide transhydrogenase is embedded in the inner membrane as a homodimer of monomer Mr = 109,288. Its N-terminal 430 residues and C-terminal 200 residues protrude into the matrix, whereas its central 400 residues appear to intercalate into the inner membrane as 14 hydrophobic clusters of about 20 residues each (Yamaguchi, M., and Hatefi, Y. (1991) J. Biol. Chem. 266, 5728-5735). Treatment of mitoplasts (mitochondria denuded of outer membrane) with several proteolytic enzymes cleaves the transhydrogenase into a 72-kDa N-terminal and a 37-kDa C-terminal fragment. The cleavage site of proteinase K was determined to be Ala690-Ala691, which is located in a small loop of the transhydrogenase exposed on the cytosolic side of the inner membrane. This paper shows that the bisected transhydrogenase can be purified from proteinase K-treated mitoplasts with retention of greater than or equal to 85% transhydrogenase activity. The inactivation rate of the bisected enzyme by trypsin and N-ethylmaleimide was altered in the presence of NADP and NADPH, suggesting substrate-induced conformation changes similar to those reported previously for the intact transhydrogenase. Also, like the intact enzyme, proteoliposomes of the bisected transhydrogenase were capable of membrane potential formation and internal acidification coupled to NADPH----NAD transhydrogenation. The properties of the bisected transhydrogenase have been discussed in relation to those of the two-subunit Escherichia coli transhydrogenase, the bisected lac permease (via gene restriction), and the fragmented and reconstituted bacteriorhodopsin.
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PMID:Mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase. Purification and properties of the proteinase K-bisected enzyme. 165 21

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.
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PMID:Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes. 193 78

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. 200 10

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23-98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23-98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23-231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.
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PMID:Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein. 1699 Feb 74

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders caused by an infectious agent termed a prion, which can convert normal cellular prion protein (PrP(C)) into a pathologically misfolded isoform (PrP(Sc)). Taking advantage of protein misfolding cyclic amplification (PMCA), a series of experiments was conducted to investigate the possible influences of pyridine nucleotides on the propagation activities of hamster-adapted scrapie agents 263K and 139A in vitro using normal hamster brain homogenates and recombinant hamster PrP as the substrates. The results showed that PrP(Sc) from both scrapie agent 263K- and 139A-infected brains propagated more efficiently in PMCA with the addition of reduced NADPH, showing an obvious dose-dependent enhancement. Reduced NADH also prompted PrP(Sc) propagation, whereas NADP, NAD and vitamin C failed. Moreover, following incubation with NADPH, recombinant hamster PrP could be efficiently converted into the proteinase K-resistant form when exposed to the trace of PrP(Sc) from infected hamsters. Our data provide evidence that the reduced pyridine nucleotide plays an important role in the propagation of prion and this process seems to target PrP(C) molecules.
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PMID:The propagation of hamster-adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro. 1922 Apr 59