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1.
Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector 总被引:1,自引:0,他引:1
E I Ginns M Rehavi B M Martin M Weller K L O'Malley M E LaMarca C G McAllister S M Paul 《The Journal of biological chemistry》1988,263(15):7406-7410
A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes. 相似文献
2.
Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level. 相似文献
3.
The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme. 相似文献
4.
P F Fitzpatrick 《Biochemistry》1991,30(26):6386-6391
Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, utilizing a tetrahydropterin and molecular oxygen as cosubstrates. Several approaches were taken to examining the identity of the rate-limiting step in catalysis. Steady-state kinetic parameters were determined with a series of ring-substituted phenylalanines. The Vmax value was unchanged with substrates ranging in reactivity from tyrosine to 4-fluorophenylalanine. Neither 4-pyridylalanine N-oxide, a model of tyrosine phenoxide, nor 4-hydroxy-3-pyridylalanine N-oxide or alpha-amino-3-hydroxy-4-pyridone-1- propionic acid, models of a hydroxycyclohexadienone intermediate, was an effective inhibitor. There was no solvent isotope effect on either the Vmax or the V/KTyr value. These results establish that no chemistry occurs at the amino acid in the rate-limiting step and no exchangeable proton is in flight in the rate-limiting step. The results are consistent with a model in which the slow step in catalysis is formation of the hydroxylating intermediate. 相似文献
5.
Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity. 总被引:1,自引:0,他引:1
S C Daubner C Lauriano J W Haycock P F Fitzpatrick 《The Journal of biological chemistry》1992,267(18):12639-12646
Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines. 相似文献
6.
M F Mineeva N R Bulatova A V Val'dman 《Biulleten' eksperimental'no? biologii i meditsiny》1990,109(4):327-330
It has been established that rat peritoneal macrophages possess tyrosine hydroxylase activity which has been characterized by KM4, 2 microM, Vmax 30 nmole/min per mg of protein. After 15 days of electronociseptive stimulation almost all tyrosine hydroxylase activity has been established in the form which has tyrosine KM47.0 M and Vmax 18.6 nmole/min. per mg of protein. Immunostimulator hydroxymethacil++, at its intraperitoneal injections to stressed rats during 7 days induced the appearance of low-affinity form of tyrosine hydroxylase with KM270 microM and Vmax 27.8 nmole/min.per mg of protein. The same low affinity form of the enzyme has been established after injections of tuftsin which possesses immunostimulating properties. 相似文献
7.
A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots (immunoblots). 相似文献
8.
Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide 总被引:8,自引:0,他引:8
Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development. 相似文献
9.
Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. 总被引:12,自引:7,他引:5
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Y M Zhang E P Hayes T C McCarty D R Dubois P L Summers K H Eckels R M Chanock C J Lai 《Journal of virology》1988,62(8):3027-3031
We have constructed a recombinant baculovirus containing a 4.0-kilobase dengue virus cDNA sequence that codes for the three virus structural proteins, capsid (C) protein, premembrane (PreM) protein, and envelope glycoprotein (E), and nonstructural proteins NS1 and NS2a. Infection of cultured Spodoptera frugiperda cells with this recombinant virus resulted in the production of E and NS1 proteins that were similar in size to the corresponding viral proteins expressed in dengue virus-infected simian cells. Other dengue virus-encoded proteins such as PreM and C were also synthesized. Rabbits immunized with the dengue virus protein products of the recombinant virus developed antibodies to PreM, E, and NS1, although the titers were low, especially to PreM and E. Nevertheless, the dengue virus antigens produced by the recombinant virus induced resistance in mice to fatal dengue encephalitis. 相似文献
10.
CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V(max) value of 1352+/-159 nmol CDP-choline/min/mg, a K(m) value of 0.50+/-0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72+/-0.10 mM for CTP. The 6 x -His-tagged enzyme has similar properties with a V(max) value of 2254+/-253 nmol CDP-choline/min/mg, a K(m) value of 0.63+/-0.13 mM for phosphocholine and a K' for CTP equal to 0.81+/-0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators. 相似文献
11.
Based on the information of molecular biology of Autographa californica Nuclear Polyhedrosis virus (AcNPV), a recombinant transfer plasmid pAcMV was constructed by molecular procedures included using two synthetic localized probes, which provided an inserted position linked with BamHI sequences nearly at polyhedrin initiating ATG codon. Then an expression vector pAcMV-HBsAg was reconstructed, it contained HBsAg gene from subclone pYPSS-1 derived from adwserotype of HBV. The recombinant virus containing HBsAg gene was isolated and purified through 3 cycles plaques and hybridization experiment after cotransfection of Spodoptera frugiperda cells with DNA of pAcMV-HBsAg and AcNPV. The expression of HBsAg gene in S. frugiperda cells infected with recombinant virus AcRV-HBsAg was identified by ELISA as haemagglutination tests. The yield of HBsAg excreted from S. frugiperda cells (an appropriate density usually between 1-2 X 10(6) cells/ml) after 48-72 h infected with AcRV-HBsAg was 4-8 mg/L. HBsAg harvested from the infected culture medium was shown immunoelectromicroscopy to be composed of spherical particles of about 22 nm diameter. Using this purified HBsAg, Bal b/c mice was immunized, the titer of anti-HBsAg serum measured measured by RIA was similar to that of purified HBsAg from human blood. Stable recombinant virus was isolated and could be shown to replicate in corn borer (Ostrinia nubilalis) larvae. All of these results can be expected that this expression vector system will be commercially developed to its fullest potential for diagnosis and vaccine HBsAg. 相似文献
12.
人尿激酶原(pro-urokinase,pro-UK)是一种新型溶栓剂,优于尿激酶,具有血纤维蛋白特异性。为了在昆虫杆状病毒表达系统中高效表达pro-UK,我们在pAc373基础上,插入野生型AcMNPV polyhedrin启动子区-7~1碱基序列,构建了一个高表达转移载体pAcYT。分别经三次克隆将pro-UK cDNA正向插入到转移载体pAc373或PAcYT的BarnHI-KpnI位点上。用LiPofectin将pAcyT-UKDNA或pAc373-UK DNA与AcMNPV DNA共转染到昆虫Sf9细胞中,空斑法筛出重组病毒阳性克隆株。高效表达结果是:1.ELISA法确定重组病毒Sf9细胞分泌表达产物pro-UK为96mg/L培养基上清,平板法测定溶圈活性为1600IU/mL培养基上清;2.亲和层析一步法纯化表达产物,回收率选70%,以上,比活约为60000IU/mg;3.纯化的pro-UK,无论是否经还原处理,其SDS-PAGE图谱均为相同的单一条带,MR 50000;4.Western blot与SDS-PAGE图谱吻合。 相似文献
13.
A cDNA coding for ovine prostaglandin endoperoxide (PGH) synthase-1 was used to construct a recombinant baculovirus which was expressed in Spodoptera frugiperda (Sf9) insect cells. Two proteins reactive with anti-PGH synthase antibody were produced. A larger protein (Mr = 72,000) coelectrophoresed with native enzyme; a smaller, more abundant protein (Mr = 66,000) was unglycosylated enzyme. About 90% of both the immunoreactivity and the cyclooxygenase activity were present in a low speed (10(5) g x min) pellet; variable but low peroxidase activities were observed in this fraction. The specific cyclooxygenase activity of solubilized PGH synthase-1 from Sf9 cells was 56 units/mg versus 112 units/mg for the same cDNA expressed in cos-1 cells. The baculovirus-insect cell system is not ideal for generating large amounts of active PGH synthase-1 apparently because of inefficient N-glycosylation. 相似文献
14.
A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells. 相似文献
15.
Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues. 相似文献
16.
Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus 总被引:1,自引:0,他引:1
A Asseffa S J Smith K Nagata J Gillette H V Gelboin F J Gonzalez 《Archives of biochemistry and biophysics》1989,274(2):481-490
Rat P450 IIA1 is a membrane-bound hemoprotein that catalyzes the 7 alpha- and 6 alpha-hydroxylation of testosterone. A recombinant baculovirus, containing the cDNA encoding IIA1, was constructed and used to infect Spodoptera frugiperda cells. Infected cells contained up to 10% IIA1 protein as quantified by Western blot analysis; however, only a small percentage of this protein contained heme and was catalytically active. Addition of hemin to the culture medium during the course of viral infection resulted in a marked sixfold increase in both testosterone hydroxylase activity and heme-containing IIA1 protein. When the exogenously added protoheme was substituted with modified heme molecules containing hydrogen or ethyl groups at the 2 and 4 positions of the protoporphyrin IX, enzymes with only marginal changes in catalytic properties were produced. These data indicate that baculovirus can be used as a system to produce high levels of mammalian cytochrome P450s containing custom modified heme moieties. 相似文献
17.
To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE-Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro. 相似文献
18.
Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. 总被引:19,自引:4,他引:19
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The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity. 相似文献
19.
B Nakhai P Sridhar G P Talwar S E Hasnain 《Indian journal of biochemistry & biophysics》1991,28(4):237-242
A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active. 相似文献
20.
Tyrosine hydroxylase phosphatase activity in rat caudate nucleus was separated into three peaks by chromatography on DEAE-cellulose. [32P]Tyrosine hydroxylase phosphorylated by cyclic AMP-dependent protein kinase was dephosphorylated only by the major peak eluting at 0.3 M NaCl, while tyrosine hydroxylase phosphorylated by Ca2+-calmodulin-dependent protein kinase was also dephosphorylated by two calcium-inhibited phosphatases. The Vmax of the enzyme in the major DEAE peak was increased by 10 microM tetrahydrobiopterin (BH4) from 0.78 to 5.0 fmol min-1 mg-1 while the Km was only slightly affected, increasing from 45 to 62 pM. The activation could not be reversed by dilution. On Sephadex G-200, the enzyme was found to consist of two major forms with molecular masses of 420 and 100 kDa. In contrast to the activation of liver phosphatases by freezing with beta-mercaptoethanol, activation by tetrahydrobiopterin was not associated with a shift in the molecular weight of the phosphatase to lower molecular weight forms. Other reduced pterins, including tetrahydroneopterin, 6-methyltetrahydropterin, and 5-methyltetrahydrofolate, also activated the enzyme, while oxidized pterins had no effect. GTP, the metabolic precursor of tetrahydrobiopterin, was a potent inhibitor of the phosphatase reaction, inhibiting by 65% at a concentration of 1 microM. These findings suggest a close regulatory interrelationship between the tetrahydrobiopterin synthetic pathway and catecholamine biosynthesis. 相似文献