Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.64 (MPP)
 1,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.
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PMID:Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization. 132 90

(3-si,4-re)-2,5-Dihydroxyacetanilide epoxidase (DHAE I), a key enzyme in the biosynthesis of the epoxysemiquinone antibiotic LL-C10037 alpha by Streptomyces LL-C10037 [Gould, S.J., & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686], and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase (DHAE II) isolated from Streptomyces MPP 3051--which yields the (3R,4S)-epoxyquinone mirror image product of DHAE I--are described. DHAE I was purified 640-fold. Gel permeation chromatography indicated an Mr of 117,000 +/- 10,000; SDS-PAGE gave a major band of 22,300 daltons, indicating that DHAE I is either a pentamer or hexamer in solution. The enzyme had a pH optimum of 6.5, a Km of 8.4 +/- 0.5 microM, and a Vmax of 3.7 +/- 0.2 mumol min-1 mg-1. DHAE II was purified 1489-fold. The enzyme was shown to be a dimer of Mr 33,000 +/- 2000, with 16,000-dalton subunits, with a pH optimum of 5.5 and a Km of 7.2 +/- 0.4 microM. Both enzymes required only O2 and substrate; flavin and nicotinamide coenzymes had little or no effect. Neither catalase nor EDTA affected the activity of either enzyme, but complete inhibition of both was obtained with 1,10-phenanthroline. The activity of the purified DHAE I could be enhanced, but only by Mn2+ (relative V = 246 at 0.04 mM), Ni2+ (relative V = 266 at 0.2 mM), or Co2+ (relative = 498 at 0.2 mM). Reconstitution from a DHAE I apoenzyme, generated by treatment with 1,10-phenanthroline followed by Sephadex G-25 chromatography, occurred only by addition of one of these three metals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Opposite facial specificity for two hydroquinone epoxidases: (3-si,4-re)-2,5-dihydroxyacetanilide epoxidase from Streptomyces LL-C10037 and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase from Streptomyces MPP 3051. 189 11

The kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying out initial velocity studies. Ca2+ and Rh(H2O)4(methylenediphosphonate) (Rh(H2O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H2O)4PP to the substrate site and Mg2+ to the "low affinity" activator site on the enzyme. Competitive inhibition was observed for Ca2+ vs Mg2+ (Kis = 0.93 +/- 0.03 mM), for Rh(H2O)4PCP vs Cr(H2O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H2O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H2O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H2O)4PP binding precedes Mg2+ ion binding is proposed. The inert substrate analog, Mg(imidodiphosphate) (MgPNP) was shown to induce Mg2+ inhibition of the PPase-catalyzed hydrolysis of MgPP. The Mg2+ inhibition observed was competitive vs MgPP and partial. These results suggest that Mg2+/MgPNP release from the enzyme occurs in preferred rather than strict order and that the Mg2+/MgPP-binding steps are at steady state. Zn2+, Co2+, and Mn2+ (but not Mg2+) displayed activator inhibition of the PPase-catalyzed hydrolysis of PPi (this study) and of Cr(H2O)4PP (W.B. Knight, S. Fitts, and D. Dunaway-Mariano, (1981) Biochemistry 20, 4079). These findings suggest that cofactor release from the low affinity cofactor site on the enzyme must precede product release and that Zn2+, Mn2+, and Co2+ (but not Mg2+) have high affinities for the cofactor sites on both the PPase.M.MPP and PPase.M.M(P)2 complexes. The role of the metal cofactor in determining PPase substrate specificity was briefly explored by testing the ability of the Mg2+ complex of tripolyphosphate (PPPi) (a substrate for the Zn2+-activated enzyme but not the Mg2+-activated enzyme) to induce Mg2+ inhibition of PPase-catalyzed hydrolysis of MgPP. MgPPP was shown to be as effective as MgPNP in inducing competitive Mg2+ inhibition (vs MgPP). This result suggests that the low affinity Mg2+ cofactor-binding site present in the enzyme-MgPP complex is maintained in the enzyme-MgPPP complex. Thus, failure of Mg2+ to bind to the enzyme-MgPPP complex was ruled out as a possible explanation for the failure of the Mg2+-activated enzyme to catalyze the hydrolysis of MgPPP.
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PMID:The kinetic mechanism of yeast inorganic pyrophosphatase. 282 96

Isolated Golgi apparatus membranes from the germinal elements (spermatocytes and early spermatids) of rat testis were examined for their ability to incorporate [14C]mannose and [14C]galactose into glycolipid and glycoprotein fractions. Transfer of mannose from GDP-[14C]mannose into a Lipid I fractions (GPD:MPP mannosyl transferase activity), identified as mannosyl phosphoryl dolichol, showed optimal activity at 1.5 mM manganese and at pH 7.5. Low concentrations of Triton X-100 (0.1%) stimulated transferase activity in the presence of exogenous dolichol phosphate (Dol-P); however, inhibition occurred at Triton X-100 concentrations greater than 0.1%. Maximal activity of this GDP:MPP mannosyl transferase occurred at 25 microM Dol-P. Activity using endogenous acceptor was 2.34 pmole/min/mg, whereas in the presence of 25 microM Dol-P the specific activity was 284 pmole/min/mg, a stimulation of 125-fold. Incorporation of mannose into a Lipid II (oligosaccharide pyrophosphoryl dolichol) and a glycoprotein fraction was also examined. In the absence of exogenous Dol-P, rapid incorporation into Lipid I occurred with a subsequent rise in Lipid II and glycoprotein fractions suggesting precursor-product relationships. Addition of exogenous Dol-P to galactosyl transferase assays showed only a minor stimulation, less than twofold, in all fractions. Over the concentration range of 9.4 to 62.5 micrograms/ml Dol-P, only 1% of radioactive product accumulated in the combined lipid fractions. These observations suggest that the mannose transfer involves Dol-P intermediates and also that spermatocyte Golgi membranes may be involved in formation of the oligosaccharide core as well as in terminal glycosylations.
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PMID:Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes. 617 52

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly149 to Arg) in the essential yeast gene CDC1. The mutant could be suppressed by overexpression of the SMF1 gene, which was isolated as an extragenic high-copy suppressor. The SMF1 gene codes for a highly hydrophobic protein and its deletion renders the yeast cells sensitive to low manganese concentration. In accordance with this observation, the smf1 null mutant exhibits reduced Mn2+ uptake at micromolar concentrations. Using a specific antibody, we demonstrated that Smf1p is located in the yeast plasma membrane. These results suggest that Smf1p is involved in high-affinity Mn2+ uptake. This assumption was also tested by overexpressing the SMF1 gene in the temperature-sensitive mutant of the mitochondrial processing peptidase (MAS1). SMF1 overexpression as well as addition of 1 mM Mn2+ to the growth medium complemented this mutation. This also suggests that in vivo Mas1p is a manganese-dependent peptidase. The yeast Smf1p resembles a protein from Drosophila and mammalian macrophages. The latter was implicated in conferring resistance to mycobacteria. A connection between Mn2+ transport and resistance or sensitivity to mycobacteria is discussed.
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PMID:A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria. 864 35

Processing of nuclear-encoded precursor proteins by mitochondrial processing peptidase (MPP) is an essential step for their sorting and function in mitochondria. We report spectroscopic studies on the catalytic mechanism of Neurospora crassa MPP. It is a complex enzyme consisting of two different subunits termed alpha-and beta-MPP. Following changes in the protein intrinsic fluorescence we register and characterize a complex formation between (i) the alpha- and the beta-subunit of MPP, (ii) the two subunits and a precursor protein, and (iii) the two subunits and some metal ions. The presequence of the precursor protein was absolutely necessary for its binding to MPP subunits. Mn2+ ions in concentrations enhancing the processing activity did not influence the substrate binding, whereas EDTA in concentrations inhibiting the enzyme completely abolished the binding of the substrate to the MPP subunits. Both MPP subunits bind metal ions such as Mn2+, Mg2+, and Zn2+. beta-MPP interacts stronger with these ions but alpha-MPP-Mn2+ conjugates seem to be important for the processing activity.
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PMID:Catalytic mechanism of mitochondrial processing peptidase: fluorescence studies. 880 41

The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation. The holoenzyme exhibited a Km value of 1.35 microM and a Vmax value of 0.25 microM/min and was inhibited by metal chelators in a time-dependent manner. Measurement of the metal content showed that both, MPP and beta-MPP, contained 0.86 and 1.05 atoms of Zn2+ per molecule, respectively. An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in beta-MPP retained less than 0.2 atom of Zn2+ per molecule. A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity. Incubation of the apoenzyme with 50 nM and 500 nM Zn2+ restored 50% and 80% of the processing activity, respectively. However, no reactivation occurred at concentrations of Zn2+ higher than 1 microM. Addition of 500 nM Mn2+ or higher concentrations (up to 50 microM) reactivated only 50% of the processing activity. The holoenzyme was competitively inhibited by molar excess of Zn2+ (Ki of 3.1 microM) but not by molar excess of Mn2+. Taken together, our data suggest that the authentic MPP is a Zn2+ rather than a Mn2+ metallopeptidase.
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PMID:The mitochondrial processing peptidase behaves as a zinc-metallopeptidase. 965 44

No mitochondrial processing peptidase (MPP) activity is detected in crystalline bovine heart mitochondrial cytochrome bc1 complex, which possesses full electron transfer activity. However, when the complex is treated with increasing concentrations of Triton X-100 at 37 degreesC, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mM of Triton X-100. This result supports our suggestion that the lack of MPP activity in the mammalian cytochrome bc1 complex is because of binding of an inhibitor polypeptide to the active site of MPP located at the interface of core subunits I and II. This suggestion is based on the three-dimensional structural information for the bc1 complex and the sequence homology between subunits of MPP and the core subunits of the beef complex. Triton X-100, at concentrations that disrupt the structural integrity of the bc1 complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to the active site of MPP in core subunits, thus activating MPP. The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temperature-dependent. Maximum activity is observed with an assay mixture containing 15 mM Tris-HCl buffer at neutral pH (6.5-8.5) and at 37 degreesC. Activated MPP is completely inhibited by metal ion chelators such as EDTA and o-phenanthroline and partially inhibited by myxothiazol (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion chelator-inhibited activity can be partially restored by the addition of divalent cations such as Zn2+ (68%), Mg2+ (44%), Mn2+ (54%), Co2+ (62%), and Fe2+ (92%), indicating that metal ion is required for MPP activity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residues of its presequence is used as a substrate.
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PMID:Activation of a matrix processing peptidase from the crystalline cytochrome bc1 complex of bovine heart mitochondria. 969 18

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non-familial Parkinson's disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP(+) (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP(+) and the other PD-related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H(2)O(2)-related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP(+), the neurotoxicants and an oxidant, H(2)O(2) equally induce the ROS-dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase-1 and Caspase-3-like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan-caspase inhibitor Boc-(Asp)-fmk (BAF) exerted significant neuroprotection against ROS-induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase-1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.
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PMID:Dopaminergic cell death induced by MPP(+), oxidant and specific neurotoxicants shares the common molecular mechanism. 1118 20

Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.
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PMID:Subtoxic concentration of manganese synergistically potentiates 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells. 1253 85


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