Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL-containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A-site, the CXXCH motif, and the N- and C-terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c-CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane-binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.

Domain swapping is an exception to Anfinsen's dogma, and more than one structure can be produced from the same amino acid sequence by domain swapping. We have previously shown that myoglobin (Mb) can form a domain-swapped dimer in which the hinge region is converted to a helical structure. In this study, we showed that domain-swapped dimerization of Mb was achieved by a single Ala mutation of Gly at position 80. Multiple Ala mutations at positions 81 and 82 in addition to position 80 facilitated dimerization of Mb by stabilization of the dimeric states. Domain swapping tendencies correlated well with the helical propensity of the mutated residue in a series of Mb mutants with amino acids introduced to the hinge region. These findings demonstrate that a single mutation in the hinge loop to modify helical propensity can control oligomer formation, providing new ideas to create high-order protein oligomers using domain swapping.[a] S.

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL‐containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A‐site, the CXXCH motif, and the N‐ and C‐terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane‐binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), n-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr- Materials Trp (tPTW), were obtained by screening of synthetic peptides forBovine pancreatic trypsin and chymotrypsin, bovine plasma thromgrowth-inhibitory activity toward cultured transformed cells. The bin, bovine plasma plasmin, porcine pancreastic elastase, human kideffects of these peptide compounds on proteases were investigated ney urokinase, bovine spleen cathepsin B, papain, and porcine kidney and the results showed that these compounds enhanced the leucine aminopeptidase (LAPase)were purchased from Sigma Chemiamidolytic activity of serine proteases despite the fact that each cal Co. (St. Louis, MO). Porcine erythrocyte m-and ~t-calpains were reaction was carried out under optimal conditions. ERPm obtained from Nakarai Chemicals (Kyoto, Japan). The substrates of proteases were obtained from Peptide Institute Inc. (Osaka, Japan). stimulated the activities of trypsin, chymotrypsin, thrombin, Peptide compounds were synthesized by conventional liquid phase plasmin urokinase and elastase, dPTWm also showed similar fragment condensation and purified as described previously [1]. The effects except that toward chymotrypsin, tPTW elevated the purity of each peptide was analyzed by thin-layer or high-performance activity only of trypsin, chymotrypsin and thrombin. Stimulation liquid chromatography. NMR spectroscopy, elemental analysis and of trypsin activity by these compounds was also confirmed by specific rotation. All peptides were dissolved in dimethylsulfoxide using casein as a substrate. None of these compounds affected the (DMSO) at a concentration of 20 mg/ml. amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m-and ~t-calpains, cathepsin B and papain) or an exopeptidase 0eucine aminopeptidase). The Assay forprotease activityAmidolytic activities of serine and cysteine proteases were measured activation was at least partly due to the stabilization of the by incubation at 37°C for 10 min in the following reaction mixture catalytic activity of proteases as well as prevention of autolysis.(0.1 ml); trypsin (100 ng), 1 x PBS, 1 mM EDTA and 10 ~tM Boc-PheSer-Arg-MCA; chymotrypsin (25 ng), 1 XPBS and 10 ~tM Suc-LeuKey words." Serine protease; Protease-stimulatory tripeptide;Leu-Val-Tyr-MCA; thrombin (1.1 ng), 50 mM Tris-HC1 (pH 7.5), 150 Synthetic tripeptide mM NaC1, 2.5 mM CaC12 and 10 laM Boc-Val-Pro-Arg-MCA; plasrain (20 lag), 1 x PBS and 10 laM Boc-Val-Leu-Lys-MCA; elastase (15 /.tg), 50 mM Tris-HC1 (pH 8.8), 1 mM EDTA and 10 laM Suc-AlaPro-Ala-MCA; urokinase (1 munit), 1XPBS, 1 mM EDTA and 10 1. Introduction gM Pyr-Gly-Arg-MCA; cathepsin B (10 launits), 50 mM MES (pH 6.0), 2 mM EDTA, 2 mM DTT and 10 ~tM Z-Arg-Arg-MCA; papain (2.5 ng), 50 mM MES (pH 5.5), 2 mM EDTA, 2 mM DTT and 10 We have previously reported the results of screening of biolaM Z-Phe-Arg-MCA; LAPase (100 launits), 50 mM Tris-HC1 (pH active peptides for growth-inhibitory activity toward trans-7.4), 5 m...

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