Effect of the SH Group of Myelopeptide MP-3 on Its Macrophage Stimulating Activity

Peptide Leu-Val-Cys-Tyr-Pro-Gln, identical to the bone marrow peptide MP-3, and its Val³and Ser³analogs, lacking SH group, were synthesized by conventional methods of peptide chemistry in solution and, along with the MP-3 S–S-dimerization product, were studied with respect to their effect on the macrophage phagocytic activity. It was shown that the activity was only enhanced by peptide MP-3, which demonstrated the essential role of the SH group in this function. The dimer analog of MP-3, unlike dimer analogs of other monocycteine-containing peptides, glutathione and HP5b, did not exhibit the inhibitory effect.     

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1β. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1β was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.     

Myelopeptide MP-5 and fluorescent derivatives: Synthesis and biological activity

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60–84% inhibition of growth of the following mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lymphocytes, but Ftc-MP-5 was practically inactive.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

Isolation and partial characterization of membrane protein constituents of human neutrophil receptors for chemotactic formylmethionyl peptides

Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.     

Influence of the bilayer composition on the binding and membrane disrupting effect of Polybia-MP1, an antimicrobial mastoparan peptide with leukemic T-lymphocyte cell selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.     

Synthesis and properties of the retro-analogue of myelopeptide MP-2

The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70-82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles.

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.     

Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).     

Structural characterization and anti-fatigue activity of polysaccharides from the roots of Morinda officinalis

Three polysaccharides MP-1, MP-2, and MP-3 were isolated from hot water extract of Chinese medicine Morinda officinalis through 95% ethanol precipitation and gel-filtration chromatography (DEAE-Sepharose CL-6B column and Sephadex G-75 or G-100 column). MP-1 was identified as an inulin-type fructan with simple linear (2 → 1)-linked structure. Both MP-2 and MP-3 were acidic polysaccharides which consisted predominantly of galacturonic acid, arabinose and galactose. Partial structure characterization of MP-3 was carried out by partial acid hydrolysis and periodate oxidation. The total polysaccharides of the herb were tested in mice weight-loaded swimming model and were found to have anti-fatigue activity.     

Fluorescently labeled differentiating myelopeptide-4: Specific binding to and penetration into target cells

Myelopeptide-4 (MP-4) (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), inducing the terminal differentiation of HL-60 leukemia cells, was labeled with fluorescein isothiocyanate. The specific binding of this modified peptide to the surface of HL-60 cells and its ability to penetrate into the cells were studied. It was shown by cytometry and confocal microscopy to be bound on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. These phenomena are probably necessary for the exhibition of MP-4 differentiating activity.     

Spatial structure of myelopeptides: I. Conformational analysis of MP-1, MP-2, and MP-3

Theoretical conformational analysis was used to study the spatial structure and conformational properties of myelopeptides, bone-marrow peptide mediators. The low-energy conformations of three hexapeptides MP-1 (Phe-Leu-Gly-Phe-Pro-Thr), MP-2 (Leu-Val-Val-Tyr-Pro-Trp), and MP-3 (Leu-Val-Cys-Tyr-Pro-Gln) were found, the values of dihedral angles of the backbone and side chains of the amino acid residues constituting these peptides were determined, and the energies of intra- and interresidual interactions were estimated.     

Effect of oligomerization on the properties of essential SH-groups of mitochondrial creatine kinase

The properties of creatine kinase isolated from bovine heart mitochondria in dimeric (Mr = 84 +/- 6 kD) and octameric (Mr = 340 +/- 17 kD) forms were compared with those of the earlier described hexameric form of the enzyme (Mr = 240 +/- 12 kD). The kinetics of SH-group modification by DTNB, the inactivation kinetics as well as the number of modified SH-groups point to significant differences between the three oligomeric forms of the enzyme. Each subunit of creatine kinase was found to possess one "fast" essential cysteine residue whose modification by DTNB and iodoacetamide led to enzyme inactivation. The formation of an analog of the transition state complex (E--MgADP--NO3--creatine) was paralleled with partial protection of only the "fast" cysteine residue which manifested itself in the decrease of the rate of its interaction with DTNB in all the three oligomeric forms. Dimer association into a hexamer and octamer occurred in parallel with a decrease of the affinity of essential SH-groups of cysteine for DTNB in 50% of the oligomeric molecule subunits. Thus, in the dimer two essential SH-groups were rapidly modified by DTNB at the same rate: k1 = k2 = (23.9 +/- 5.6).10(4) M-1 min-1. Within the hexamer, the rate of modification of 3 out of 6 SH-groups was practically unchanged: k1 = (10.6 +/- 2.3).10(4) M-1 min-1. Another 3 SH-groups in the remaining 50% of the subunits were partly masked, which manifested itself in a 10-fold decrease of their modification rate: k2 = (1.12 +/- 0.28).10(4) M-1 min-1. Within the octamer, the SH-groups rapidly interacted with DTNB only on 4 subunits: k1 = (20.7 +/- 2.2).10(4) M-1 min-1, whereas in the remaining 4 octamer subunits a practically complete masking of essential SH-groups was observed, as a result of which these groups became inaccessible to DTNB. This manifested itself in a 1000-fold decrease of the rate of SH-group modification by DTNB which reached that of non-essential SH-group modification. In has been found that a complete loss of the octamer activity is due to the modification of only 4 SH-groups which interact with DTNB at a high rate. A model for subunit association into a dimer, hexamer and octamer has been proposed. Presumably, 50% of the active centers in the mitochondrial creatine kinase octamer are not involved in the catalytic act.     

Identification and Characterization of Calcium-Dependent Metalloproteases in Rat Brain

We have begun to identify and characterize brain protease activities separated by and assayed in substrate-containing polyacrylamide gels. In the present report, we focus on four proteolytic activities identified from rat brain that are dependent on micromolar and millimolar Ca2+ concentrations for activity. In contrast to the previously described Ca2+-dependent neutral cysteine proteases (calpains), all four activities appear to be metalloproteases based on their inhibition by EDTA, EGTA, and 1,10-o-phenanthroline, but not by blockers of serine, cysteine, or aspartic proteases. In the presence of excess Ca2+ and the Zn2+-chelating inhibitor 1,10-o-phenanthroline, activity of the enzymes was reconstituted by addition of lower concentrations of Zn2+, and inhibited by higher Zn2+ concentrations. The four metalloproteases were designated MP-112, MP-92, MP-70, and MP-65 on the basis of their apparent molecular masses in kilodaltons. MP-70, the major activity detected, had an apparent kact for Ca2+ greater than 100 microM versus 10-25 microM for MP-65 and 50-100 microM for MP-92. MP-112 was a minor activity for which Ca2+ activation levels were not determined. MP-112, MP-70, and MP-65 were similar in being most active in the soluble fraction of 7-day neonate forebrain. In contrast, MP-92 activity was highest in the particulate fraction of adult forebrain. About half of the MP-92 activity and lower levels of the other three activities were still detectable in particulate fractions after detergent extraction of membrane, suggesting an association with cytoskeletal or other structural proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

抗HIV-1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响(英)

研究了具有抗HIV-1活性的大环多胺类化合物与RNA的识别作用，以及其对cos-7细胞凋亡的影响，以进一步探讨其抗HIV-1的作用机理.实验采用琼脂糖凝胶电泳方法, 观察化合物与RNA的识别作用；通过流式细胞计数法探讨其对cos-7细胞凋亡的影响；运用计算机分子模型, 从理论上Docking计算化合物与TAR RNA结合的可能性.结果表明, 大环多胺类化合物MP-1、MP-2和MP-3不仅具有断裂RNA的作用，并可抑制Tat-RNA的相互作用, 还可影响cos-7细胞亚二倍体的含量; 理论化学计算数据与实验结果基本一致.这一结果提示化合物的抗HIV-1活性可能通过作用于病毒基因组RNA而发挥作用，是多靶作用的结果.     

Fluorescently labeled differentiating myelopeptide-4: specific binding to and penetration into target cells

Myelopeptide-4 (MP-4) (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), inducing the terminal differentiation of HL-60 leukemia cells, was labeled with fluorescein isothiocyanate. The specific binding of this modified peptide to the surface of HL-60 cells and its ability to penetrate into the cells were studied. It was shown by cytometry and confocal microscopy to be bound on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. These phenomena are probably necessary for the exhibition of MP-4 differentiating activity.     

Helix-stabilizing effects of the pentapeptide KIFMK and its related peptides on the sodium channel inactivation gate peptides.

We have previously found by NMR and CD spectroscopic studies that the helical content of the sodium channel inactivation gate-related peptide (Ac-GGQDIFMTEEQK-NH2; MP-1A) in 80% trifluoroethanol solutions was increased by adding a pentapeptide, KIFMK. In order to study in further detail whether the presence of the IFM motif and the two lysine residues is a prerequisite for stabilizing the helical conformation, we examined interactions between various oligopeptides (RIFMR, KIFMTK, KIQMK, KAFAK, KIIIK) and MP-1A and its related peptides; that is, MP-2A in which Phe was replaced by Gln, MP-1MMA in which Thr was replaced by Met, MP-1TA in which Thr was removed from MP-1A, and MP-1A' in which L-Phe was replaced by D-Phe. It was found that the IFM motif was absolutely necessary in both the oligopeptide and the inactivation gate peptide. This finding means that hydrophobic interactions are operative between KIFMK and MP-1A. In contrast, KIFMK destabilized the helical structure of MP-1MMA, MP-1TA, and MP-1A', showing that the conformation around the IFM motif in the inactivation gate peptides is an important factor. It was concluded that the IFM motif and the two Lys residues are a prerequisite for effectively stabilizing the alpha-helix of MP-1A.     

Endogenous immunoregulatory peptides (myelopeptides): structure, function, and mechanism of action

Previously unknown bioregulators from bone marrow, myelopeptides, were isolated, identified, and synthesized, and their biological properties and mechanism of action were studied in detail. Phe-Leu-Gly-Phe-Pro-Thr (MP-1) manifests an immunocorrecting effect by restoring the level of antibody production in animals suffering from immunodeficiencies of various etiologies; Leu-Val-Val-Tyr-Pro-Trp (MP-2) manifests an antitumor effect by abolishing the inhibitory action of tumor cells on the functional activity of T-lymphocytes; Leu-Val-Cys-Tyr-Pro-Gln (MP-3) stimulates phagocytosis by macrophages and, in this way, protects animals from infections caused by pathogenic microorganisms; and Phe-Pro-Arg-Ile-Met-Thr-Pro (MP-4) is a new factor of cell differentiation inducing terminal cell differentiation in the HL-60 and K-562 leukemic cell lines.     

Effect of myelopeptide-1 on the functional activity of phagocytes from mice treated with cyclophosphane

The capacity of the bone marrow-derived myelopeptide-1 (MP-1) to affect in vivo and in vitro the functional activity of phagocytes of intact mice and mice treated with a cytostatic agent (cyclophosphane) has been studied. It was found that MP-1 produces a correcting effect on the functional activity of bone marrow and peripheral blood phagocytes. An optimal scheme of the injection of MP-1 to mice with the cyclophosphane-induced immunodeficiency was developed, which provides a maximum immunocorrecting action. MP-1 had the most pronounced effect on the quantitative characteristics and the functional activity of phagocytes of different localization when introduced prior to the cytostatic; under these conditions, the pep tide affects peripheral blood neutrophils. The results obtained enable one to consider MP-1 as a preparation protecting the peripheral blood phagocytes from the damaging action of cyclophosphane.     

抗HIV—1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响

研究了具有抗HIV－1活性的大环多胺类化合物与RNA的识别作用,以及其对cos-7细胞凋亡的影响,以进一步探讨其抗HIV－1的作用机理。实验采用琼脂糖凝胶电泳方法。观察化合物与RNA的识别作用。通过流式细胞计数法探讨其对cos-7细胞凋亡的影响。运用计算机分子模型,从理论上Docking计算化合物与TAR RNA结合的可能性。结果表明,大环多胺类化合物MP－1、MP－2和MP－3不仅具有断裂RNA的作用,并可抑制Tat-RNA的相互作用,还可影响cos-7细胞亚二倍体的含量;理论化学计算数据与实验结果基本一致,这一结果提示合物的抗HIV－1活性可能通过作用于病毒基因组RNA而发挥作用,是多靶作用的结果。