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1.
Rajala V. S. Raju Raju S. S. Datla Rajendra K. Sharma 《Molecular and cellular biochemistry》1996,155(1):69-76
Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins.Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)4-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP711 a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. TheE. coli expressed hNMT was homogeneous and showed enzyme activity.Abbreviations NMT
N-myristoyl CoA:protein N-myristoyltransferase
- NIP71
71 kDa heat stable membrane bound N-myristoyltransferase inhibitor protein
- hNMT
human NMT
- DTNB
N-5,5dithiobis (2-nitrobenzoic acid)
- FPLC
fast protein liquid chromatography
- IPTG
isopropyl -D-thiogalactopyranoside
- cDNA
complementarydeoxyribonucleic acid
- SDS
sodium dodecyl sulfate
- PMSF
phenylmethylsulfonyl fluoride 相似文献
2.
Abstract: An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60src . This N -myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Mi-chaelis-Menten kinetics yielding K m values of 100 μ M and 60 μ M , and Vmax values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and sre-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N -myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord. 相似文献
3.
Rajala RV Datla RS Carlsen SA Anderson DH Qi Z Wang JH Sharma RK 《Biochemical and biophysical research communications》2001,288(1):233-239
N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the amino terminal glycine residue of a number of important proteins especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn and Lck and dephosphorylated by the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin. Deletion of 149 amino acids from the N-terminal end resulted in the absence of phosphorylation suggesting that the phosphorylation sites are located in the N-terminal end of NMT. Furthermore, a site-directed mutagenesis study indicated that substitution of tyrosine 100 with phenylalanine served NMT as a poor substrate for the Lyn kinase. A synthetic peptide corresponding to the amino-terminal region encompassing tyrosine 100 of NMT served as a good substrate for the Lyn and Fyn kinases. Our studies also indicated that NMT was found to interact with Lyn through its N-terminal end in a phosphorylation-dependent manner. This is the first study demonstrating the cross-talk between NMT and their myristoylated protein substrates in signaling pathways. 相似文献
4.
Rajala V. S. Raju Raju S. S. Datla Rajendra K. Sharma 《Protein expression and purification》1996,7(4):431-437
Myristoyl CoA:proteinN-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding humanN-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP–Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS–polyacrylamide gel electrophoresis. Antibodies toEscherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The overexpressed hNMT was homogeneous and showed enzyme activity. 相似文献
5.
GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. 相似文献
6.
Wu J Tao Y Zhang M Howard MH Gutteridge S Ding J 《The Journal of biological chemistry》2007,282(30):22185-22194
Protein N-myristoylation catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) plays an important role in a variety of critical cellular processes and thus is an attractive target for development of antifungal drugs. We report here three crystal structures of Saccharomyces cerevisiae NMT: in binary complex with myristoyl-CoA (MYA) alone and in two ternary complexes involving MYA and two different non-peptidic inhibitors. In all three structures, the majority of the N-terminal region, absent in all previously reported structures, forms a well defined motif that is located in the vicinity of the peptide substrate-binding site and is involved in the binding of MYA. The Ab loop, which might be involved in substrate recognition, adopts an open conformation, whereas a loop of the N-terminal region (residues 22-24) that covers the top of the substrate-binding site is in the position occupied by the Ab loop when in the closed conformation. Structural comparisons with other NMTs, together with mutagenesis data, suggest that the N-terminal region of NMT plays an important role in the binding of both MYA and peptide substrate, but not in subsequent steps of the catalytic mechanism. The two inhibitors occupy the peptide substrate-binding site and interact with the protein through primarily hydrophobic contacts. Analyses of the inhibitorenzyme interactions provide valuable information for further improvement of antifungal inhibitors targeting NMT. 相似文献
7.
Pittman JK Shigaki T Cheng NH Hirschi KD 《The Journal of biological chemistry》2002,277(29):26452-26459
Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism. 相似文献
8.
All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells. 相似文献
9.
Human methionine sulfoxide reductase B3A (hMsrB3A) is an endoplasmic reticulum (ER) reductase that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine in proteins. In this work, we identified an antimicrobial peptide from hMsrB3A protein. The N-terminal ER-targeting signal peptide (amino acids 1-31) conferred an antimicrobial effect in Escherichia coli cells. Sequence and structural analyses showed that the overall positively charged ER signal peptide had an Argand Pro-rich region and a potential hydrophobic α-helical segment that contains 4 cysteine residues. The potential α-helical region was essential for the antimicrobial activity within E. coli cells. A synthetic peptide, comprised of 2-26 amino acids of the signal peptide, was effective at killing Gram-negative E. coli, Klebsiella pneumoniae, and Salmonella paratyphi, but had no bactericidal activity against Gram-positive Staphylococcus aureus. 相似文献
10.
N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Little is known about the control and regulation of NMT in higher eukaryotes. Bovine spleen N-myristoyltransferase has been purified and characterized [Raju, RVS, Kalra J & Sharma RK (1994) J Biol Chem 269:12080–12083]. The activation of bovine spleen NMT with thiol reducing compounds, and its inhibition by the oxidizing agent sodium iodate, suggest a role for oxidation/reduction in NMT regulation. Available knowledge concerning coenzyme A (CoA), the thiol in the cell, indicated that the agents tested on NMT could also reduce or oxidize CoA. The studies suggested that reduced CoA is the key regulator of NMT activity, while oxidized CoA did not allow NMT to promote myristoylation. Further, the process of myristoylation and demyristoylation may be governed by NMT, depending on the differential concentration of CoA. The process of demyristoylation could be blocked by excess CoA. We therefore hypothesize that the initial event in the regulation of NMT is an increase in cellular CoA concentration which could be coupled to an increase in protein myristoylation. Once the CoA concentration in the cell decreases due to oxidation, the demyristoylation process would be operative.Abbreviations NMT
N-myristoyl CoA:protein N-myristoyltransferase
- hNMT
human NMT
- YNMT
yeast NMT
- DTNB
N-55 dithiobis(2-nitrobenzoic acid)
- DTT
dithiothretol
- 2-ME
2-mercaptoethanol 相似文献
11.
The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed. 相似文献
12.
Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. 相似文献
13.
SIRT1 is a NAD+-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD+-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD+-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain. 相似文献
14.
A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester. 总被引:13,自引:5,他引:8
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We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene. 相似文献
15.
N-terminal N-myristoylation is a lipid anchor modification of eukaryotic and viral proteins targeting them to membrane locations, thus changing the cellular function of modified proteins. Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export. The myristoyl-CoA:protein N-myristoyltransferase (NMT) recognizes the sequence motif of appropriate substrate proteins at the N terminus and attaches the lipid moiety to the absolutely required N-terminal glycine residue. Reliable recognition of capacity for N-terminal myristoylation from the substrate protein sequence alone is desirable for proteome-wide function annotation projects but the existing PROSITE motif is not practical, since it produces huge numbers of false positive and even some false negative predictions.As a first step towards a new prediction method, it is necessary to refine the sequence motif coding for N-terminal N-myristoylation. Relying on the in-depth study of the amino acid sequence variability of substrate proteins, on binding site analyses in X-ray structures or 3D homology models for NMTs from various taxa, and on consideration of biochemical data extracted from the scientific literature, we found indications that, at least within a complete substrate protein, the N-terminal 17 protein residues experience different types of variability restrictions. We identified three motif regions: region 1 (positions 1-6) fitting the binding pocket; region 2 (positions 7-10) interacting with the NMT's surface at the mouth of the catalytic cavity; and region 3 (positions 11-17) comprising a hydrophilic linker. Each region was characterized by physical requirements to single sequence positions or groups of positions regarding volume, polarity, backbone flexibility and other typical properties of amino acids (http://mendel.imp.univie.ac.at/myristate/). These specificity differences are confined partly to taxonomic ranges and are proposed for the design of NMT inhibitors in pathogenic fungal and protozoan systems including Aspergillus fumigatus, Leishmania major, Trypanosoma cruzi, Trypanosoma brucei, Giardia intestinalis, Entamoeba histolytica, Pneumocystis carinii, Strongyloides stercoralis and Schistosoma mansoni. An exhaustive search for NMT-homologues led to the discovery of two putative entomopoxviral NMTs. 相似文献
16.
UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain. 相似文献
17.
蛋白质精氨酸甲基转移酶5(PRMT5)在细胞生长和信号转导方面是一个重要的调节因子,主要参与染色质重塑、RNA剪切、基因转录、细胞分化等过程.因此,对其结构和功能的研究就显得十分重要.通过大肠杆菌表达系统把全长基因PRMT5构建到pGEX-4T-1表达载体上,所得到GST标签的重组蛋白可溶性很低.为此,通过在其N端缺失不同氨基酸序列来增加其表达量,而且其中有一个缺失突变体的活性并没有发生改变.同时,还发现PRMT5 N端的前15个氨基酸对其甲基转移酶的催化活性很重要. 相似文献
18.
The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex. 相似文献
19.
Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding. 总被引:1,自引:0,他引:1
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Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose. A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes. 相似文献
20.
The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR10AS 12 VAQLTQEL20GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme??s active site, affected the enzyme??s activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme??s main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure. 相似文献