Apolipoprotein composition and particle size affect HDL degradation by chymase: effect on cellular cholesterol efflux

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal pre beta-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase. We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.     

Species Differences in Angiotensin II Generation and Degradation by Mast Cell Chymases

Abstract

Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr⁴-Ile⁵ site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe⁸-His⁹ bond using the model peptide Ang, Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹-Leu¹⁰), the chymases ranked as follows:dog > human > hamster > mouse > rat (k_cat/K_m: 18, 11, 0.69, 0.059, 0.030 μ M^{? 1}min^{? 1}), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr⁴-Ile⁵ bond of Ang II), the order was hamster > rat > mouse > dog (k_cat/K_m: 5.4, 4.8, 0.39, 0.29 μ M^?1min^?1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.     

Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2'' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr₄-Ile₅ bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.     

Differential Cleavage of Urodilatin and Atrial Natriuretic Factor by Thrombin and Protease 3.4.24.11

Abstract

Human urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine⁹⁸ to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cys^l05 and Phe¹⁰⁶. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys¹⁰⁵-Phe¹⁰⁶ site as for ANF with a second cleavage site at Gly¹¹⁸-Leu¹¹⁹. However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF. A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed K_m values of 11.7 and 3.1 μM, respectively. The turnover rates (k_cat/K_m) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min^?1 μM^?1, respectively. Thus, urodilatin is much less efficiently hydrolyzed by purified NEP than is ANF. The four residue extension at the N-terminus of urodilatin may be important for protection against rapid biological inactivation.     

Specificities of extracellular and ribosomal serine proteinases from Bacillus natto,a food microorganism

The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Ser⁹-His¹⁰ and additional cleavage sites at Gln⁴-His⁵ and His⁵-Leu⁶ in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe⁸-His⁹ and secondary at Tyr⁴-Ile⁵. The extracellular serine proteinase has a K_m of 0.08 mM and k_cat of 3 s⁻¹ for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu¹⁵-Tyr¹⁶ and additional cleavage site at Phe²⁴-Phe²⁵. Hydrolysis of proangiotensin was observed at Tyr⁴-Ile⁵ bond with the ribosomal proteinase.     

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr¹⁶⁶), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr¹⁶⁶ nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO₂-Tyr¹⁶⁶-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNA_CUA pair for functional studies. Studies with mAb 4G11.2 showed that NO₂-Tyr¹⁶⁶-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO₂-Tyr¹⁶⁶-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063–1.21). NO₂-Tyr¹⁶⁶-apoA-I in plasma showed a similar distribution. Recovery of NO₂-Tyr¹⁶⁶-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr¹⁶⁶ is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.     

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R₁ and MCH‐R₂, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R₁ was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R₁ and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R₁ (SLC‐1), the radioligand was [¹²⁵I]–[Tyr¹³]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [¹²⁵I]–[D‐Phe¹³, Tyr¹⁹]‐MCH served as radioligand. The bioassay used for MCH‐R₁ was intracellular Ca²⁺ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R₁ and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe¹³ in [Phe¹³, Tyr¹⁹]‐MCH was well tolerated by both MCH‐R₁ and B16 MCH‐R, change of configuration to D‐Phe¹³ in [D‐Phe¹³, Tyr¹⁹]‐MCH or [D‐Phe¹³]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R₁. By contrast, the D‐Phe¹³ residue increased the affinity of [D‐Phe¹³, Tyr¹⁹]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe¹³, Tyr¹⁹]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R₁ in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R₁.     

A protein tyrosine phosphatase inhibitor,pervanadate, inhibits angiotensin II-Induced β-arrestin cleavage

β-Arrestins turn off G protein-mediated signals and initiate distinct G protein-independent signaling pathways. We previously demonstrated that angiotensin AT₁ receptor-bound β-arrestin 1 is cleaved after Phe³⁸⁸ upon angiotensin II stimulation. The mechanism and signaling pathway of angiotensin II-induced β-arrestin cleavage remain largely unknown. Here, we show that protein Tyr phosphatase activity is involved in the regulation of β-arrestin 1 cleavage. Tagging of green fluorescent protein (GFP) either to the N-terminus or C-terminus of β-arrestin 1 induced conformational changes and the cleavage of β-arrestin 1 without angiotensin AT₁ receptor activation. Orthovanadate and molybdate, inhibitors of protein Tyr phosphatase, attenuated the cleavage of C-terminal GFP-tagged β-arrestin 1 in vitro. The inhibitory effects of okadaic acid and pyrophosphate, which are inhibitors of protein Ser/Thr phosphatase, were less than those of protein Tyr phosphatase inhibitors. Cell-permeable pervanadate inhibited angiotensin II-induced cleavage of β-arrestin 1 in COS-1 cells. Our findings suggest that Tyr phosphorylation signaling is involved in the regulation of angiotensin II-induced β-arrestin cleavage.     

Substrate Specificity of a Novel Alcohol Resistant Metalloproteinase,Vimelysin, from Vibrio sp. T1800

Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin.     

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of Phe_D1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative E_m of Phe_D1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S₂Q_A⁻/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the E_m of Phe_D1^–/Phe_D1 was decreased by ∼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S₂Tyr_Z^• to S₃Tyr_Z and S₃Tyr_Z^• → (S₃Tyr_Z^•)’ transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V_max/K_m) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

To the problem of biologically active conformation of enkephalin

Summary A semi-rigid structural analog of [Leu⁵] enkephalin, possessing the azo-bridge between Tyr¹ and Phe⁴ residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe⁴] enkephalin and [4′hydroxyphenyl/-azo Phe⁴] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr¹ and Phe⁴, are situated in close proximity.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O₂-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 is biphasic for all the S-state transitions except for S₁ to S₂, and shows that: i) the proton release in S₀ to S₁ occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S₂ to S₃, in T. elongatus, are significantly faster than often considered. The nature of S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

Site-Directed Mutagenesis of Active Site Residues Reveals Plasticity of Human Butyrylcholinesterase in Substrate and Inhibitor Interactions

Abstract: In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser¹⁹⁸, the acyl pocket Leu²⁸⁶ and adjacent Phe³²⁹ residues, and Met⁴³⁷ and Tyr⁴⁴⁰ located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and K_m values towards butyrylthiocholine and IC₅₀ values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser¹⁹⁸ by cysteine and Met⁴³⁷ by aspartate nearly abolished activity, and other mutations of Ser¹⁹⁸ completely abolished it. Tyr⁴⁴⁰ and Leu²⁸⁶ mutants remained active, but with higher K_m and IC₅₀ values. Rates of inhibition by DFP were roughly parallel to IC₅₀ values for several Leu²⁸⁶ mutants. Both K_m and IC₅₀ values increased for Leu²⁸⁶ mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe³²⁹ displayed unmodified K_m values toward butyrylthiocholine, but up to 10-fold decreased IC₅₀ values for DFP, iso-OMPA, and echothiophate. These findings add Tyr⁴⁴⁰ and Phe³²⁹ to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.     

The Human Cdc34 Carboxyl Terminus Contains a Non-covalent Ubiquitin Binding Activity That Contributes to SCF-dependent Ubiquitination

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1·Cullin 1·F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206–215 and 216–225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys⁴⁸ and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe²⁰⁶, Tyr²⁰⁷, Tyr²¹⁰, and Tyr²¹¹) are probably positioned in the vicinity of ubiquitin C-terminal residue Val⁷⁰. Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val⁷⁰ by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1–215) but not Cdc34(1–200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IκBα ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe²⁰⁶/Tyr²⁰⁷, Tyr²¹⁰/Tyr²¹¹) in conjugation, with Tyr²¹⁰ exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr²¹⁰ was required for the transfer of the donor ubiquitin to a receptor lysine on either IκBα or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.     

Cleavage of Rabbit Myelin Basic Protein by Pepsin

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe⁸⁷-Phe⁸⁸ bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu¹⁵¹-Phe¹⁵² bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe⁴⁴-Phe⁴⁵ and Leu¹⁰⁹-Ser¹¹⁰ bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH₂-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr¹⁴Leu¹⁵ bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.     

Purification,characterization and substrate specificity of a basic proteinase in the venom of Habu (Trimeresurus flavoriridis)

A basic proteinase was purified and characterized from the venom of Habu (Trimeresurus flavoviridis). Its molecular weight, isoelectric point and optimum pH were approx. 24 000, 9.2 and 9, respectively. Susceptibility to several reagents was examined. The proteinase had endopeptidase activity cleaving the Gly-Leu bond in synthetic peptides but no exopeptidase activity. It did not hydrolyze a peptide, Z-Gly-Pro-Leu-Gly-Pro, which had been a good substrate for the major proteinase in the venom. The proteinase cleaved oxidized insulin B chain at five positions: His₁₀-Leu₁₁, Ala₁₄-Leu₁₅, Tyr₁₆-Leu₁₇, Gly₂₃-Phe₂₄ and Phe₂₄-Phe₂₅. From the disappearance of intermediate peptides and the peptides accumulated, the order and the intensity of cleavage of these positions were determined, and the substrate specificity was compared with those hitherto described for hemorrhagic and nonhemorrhagic venom proteinases.     

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically‐modified [Tyr¹]FQ (1‐6), [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β‐sheet conformation. FQ (1‐11), FQ (7‐17) [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) have an α‐helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ～8.3) was investigated by means of surface‐enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7‐17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1‐11), FQ (1‐6), [Tyr¹]FQ (1‐6), and [His¹]FQ (1‐6) lies almost flat on this surface, while for FQ (1‐13) and FQ (1‐13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1‐6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1‐6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1‐13) and FQ (7‐17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the ? NH₂ group. We also showed that upon adsorption, the secondary structure of these peptides is altered. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1039–1054, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The temperature dependence and thermodynamic functions of partitioning of substance P peptides in dodecylphosphocholine micelles

The temperature dependence of the partition of a neuropeptide, Substance P (SP), and its [Tyr⁸] analogue in a widely used membrane mimic, dodecylphosphocholine micelles, was studied by using a pulsed field gradient nmr diffusion technique. The partition coefficient was found to decrease when the temperature is increased, indicating a favorable (negative) enthalpy change upon partitioning of the peptides. Thermodynamic functions of the partitioning were determined. The enthalpy of partition ΔH_part, was found to be in the −2.5 to −3.0 kcal/mol range, which is between 2 and 3 times higher than the entropic term −TΔS_part. The free energy of partitioning is consistent with a model in which the SP peptides interact with the micelles mainly through the hydrophobic side chains of the residues Phe⁷, Phe⁸ (or Tyr⁸), Leu¹⁰, and Met¹¹, and without the insertion of a major portion of the peptide into the hydrophobic core of the micelles. © 1998 John Wiley & Sons, Inc. Biopoly 45: 395–403, 1998     

Substrate specificity of carboxyl proteinase ofAspergillus sojae

The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu¹⁵-Tyr¹⁶ bond and the Phe²⁴-Phe²⁵ bond. Additional cleavage of the bond Tyr¹⁶-Leu¹⁷ was also noted.