Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mutation that causes a deficiency of the lysosomal amidase, glycosylasparaginase, has been characterized in fibroblasts from three Finnish patients diagnosed with aspartylglucosaminuria (AGU). The polymerase chain reaction was used to amplify the glycosylasparaginase protein coding sequence from the three AGU patients in order to compare them to the normal sequence from a full-length human placenta cDNA clone HPAsn.6 (Fisher, K.J., Tollersrud, O.K., and Aronson, N.N., Jr. (1990) FEBS Lett. 269, 440-444). Two base changes were found to be common to all three Finnish AGU patients, a G482----A transition that results in an Arg161----Gln substitution and a G488----C transversion that causes Cys163----Ser. Detection of both point mutations from PCR-amplified cDNA or genomic DNA was facilitated by their creation of new endonuclease restriction sites. Expression studies in COS-1 cells revealed only the Cys163----Ser mutation caused a deficiency of glycosylasparaginase activity. This same substitution also prevented the normal posttranslational processing of the precursor glycosylasparaginase polypeptide into its alpha and beta subunits. Cell-free expression of the single-chain glycosylasparaginase precusor did not produce an active enzyme, suggesting that post-translational generation of subunits may be required for catalytic activity.
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PMID:Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. Amino acid substitution Cys163----Ser abolishes the activity of lysosomal glycosylasparaginase and its conversion into subunits. 190 74

Aspartylglucosaminidase (AGA, E.C. 3.5.1.26) is a soluble lysosomal hydrolase that participates in the degradation of glycoproteins. Here we analyzed the special features in the intracellular targeting of this dimeric amidohydrolase, especially the role of N-linked sugars and their phosphorylation in transport and activity of heterodimeric aspartylglucosaminidase, using in vitro mutagenesis and transient expression of mutant polypeptides in COS cells. The single N-glycosylation sites of both the alpha and beta subunits were destroyed individually and in combination. Just one remaining N-glycosylation site on either subunit was sufficient for normal processing into subunits and lysosomal transport, but the totally nonglycosylated enzyme, although active and processed into subunits, was not transported into lysosomes and became trapped in the endoplasmic reticulum (ER) or secreted. The intracellular targeting of AGA was partially disturbed by the lack of glycosylation in the beta subunit, resulting in accumulation of dimeric, active polypeptides in the ER, whereas lack of oligosaccharides in the alpha subunit did not affect the intracellular targeting of AGA. N-glycans in the beta subunit were found to be essential for the long-term stability of the polypeptide in the cell, but not for initial folding or subunit processing into the active dimeric molecule. Both subunits have two glycosylation isoforms. Both forms of the alpha subunit were found to be phosphorylated, whereas only one of the two glycosylation isoforms of the beta subunit is phosphorylated. The mutant enzyme with nonglycosylated alpha subunit and nonphosphorylated beta subunit is transported into lysosomes, suggesting that AGA is capable of using an alternative, mannose-6-phosphate receptor-independent routing into lysosomes.
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PMID:Intracellular sorting of aspartylglucosaminidase: the role of N-linked oligosaccharides and evidence of Man-6-P-independent lysosomal targeting. 771 Jun 87

1. The Brown Norway (B/N) Katholiek rat is a mutant strain of plasma kininogen deficiency. The plasma of B/N-Katholiek rats was shown to contain only 3-5% of high-molecular-weight and low-molecular-weight kininogens (HK and LK) of the normal level by specific RIA, and 30% of prekallikrein was detected by amidase activity. However, HK antigen in the liver microsomal fraction of B/N-Katholiek rats was about 60% of that of normal rats. 2. In this paper we compare and discuss synthesis and secretion of HK and LK by primary cultures of livers of deficient and normal rats. The deficient hepatocytes could synthesize HK and LK in the same way as normal cells but could not secrete mature forms of HK and LK in the medium. Examination of the subcellular localization of the mutant HK in the hepatocytes showed that a larger amount of mutant HK antigen, compared to normal rats, was found in the 10,000 g fraction, which is rich in lysosomes, suggesting that the mutant HK may be transported to the lysosomes. 3. We also analyzed sequence of the HK cDNA of B/N-Katholiek and B/N-Kitasato rats and found a point mutation of G to A at nucleotide 487, which locates at the heavy chain region of HK and LK. 4. We constructed five expression plasmids to transfect COS-1 cells to examine HK secretion. COS-1 cells transfected with the plasmids containing the G to A transition could not secrete and retained HK, while those cells transfected with the plasmids containing normal G released HK into the medium. 5. These results indicate that a point mutation G to A at nucleotide 487, resulting in an amino acid transition from alanine (163) to threonine, is responsible for the defective secretion of HK and LK by the liver of B/N-Katholiek rats. We also discuss other cases of secretion defect of plasma proteins reported in the literature.
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PMID:Molecular mechanism of kininogen deficiency in brown Norway Katholiek rats. 774 70

Aspartylglucosaminuria (AGU) is an inborn error of glycoprotein catabolism and represents the only known human deficiency of an amidase, aspartylglucosaminidase (AGA, EC 3.5.1.26). We report here a detailed characterization of a unique 2 kb deletion of the AGA gene in a North American AGU patient. To facilitate the characterization of the deletion, genomic lamda clones spanning the 3' flanking region of human AGA were isolated and sequenced. The breakpoint of the deletion was determined from the patient's DNA by sequencing the genomic region containing the novel junction. The rearrangement involved a nonhomologous recombination with only 2 bp of homology at the deletion breakpoint. The deletion's 5' breakpoint was located in the last intron of AGA, thus abolishing the normal C-terminal exon. This is in contrast to our previous findings indicating that the deletion in the AGA gene would contain only the complete 3' untranslated region and leave the coding region intact (1). The unique feature of this deletion is a triplication of 19 thymidine nucleotides of an inverted Alu repeat, which is located at the deletion 3' breakpoint. The analysis of the patient's AGA cDNA revealed an open reading frame containing a novel C-terminal exon, coding for a 64 amino acid sequence, which has no homology to the normal exon 9 of AGA. This new exon has a functional splice acceptor site at its 5' end, a stop codon, and a polyadenylation signal at the 3' end. Expression of the mutant AGA cDNA in COS cells showed that mutant mRNA is synthesized in equal amounts compared with normal.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Deletion of the C-terminal end of aspartylglucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement. 779 99

Previously we suggested that one porcine brain enzyme (anandamide amidohydrolase) catalyzed both the hydrolysis of anandamide and its synthesis from arachidonic acid and ethanolamine (Ueda et al., J. Biol. Chem. 270, 23823-23827, 1995). In the present study we investigated the reversibility of the enzyme reactions by the use of recombinant fatty-acid amide hydrolase of rat liver, which appears to be catalytically identical to porcine anandamide amidohydrolase. The particulate fraction of the COS-7 cells, in which the rat enzyme was overexpressed, hydrolyzed anandamide with a specific activity of 132 nmol/min/mg protein at 37 degrees C, and the Km value for anandamide was 18 microM. The enzyme also synthesized anandamide at a rate of 177 nmol/min/mg protein, and the Km values for arachidonic acid and ethanolamine as substrates were as high as 190 microM and 36 mM, respectively. The control cells transfected with the insert-free vector showed neither the hydrolase activity nor the synthase activity. Thus, the hydrolase and synthase are attributed to the same enzyme protein coded by one gene. However, the enzyme may act as a hydrolase rather than a synthase under physiological conditions judging from its high Km values for substrates in the synthase reactions. In addition, primary amides of fatty acids such as arachidonamide and oleamide and fatty acid ester like methyl arachidonate were hydrolyzed at considerable rates, and their reverse reactions occurred even if at lower rates.
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PMID:Reversible hydrolysis and synthesis of anandamide demonstrated by recombinant rat fatty-acid amide hydrolase. 929 94

Anandamide amidase is the hydrolytic enzyme responsible for the breakdown of anandamide, an endogenous cannabimimetic, to arachidonate and ethanolamine. Another enzymatic activity called anandamide synthase catalyzes the reverse reaction, that is the condensation of arachidonate and ethanolamine. Using a recently cloned rat fatty acid amidohydrolase (FAAH), we tested the hypothesis that the synthase and the amidase activities are catalyzed by the same enzyme. Untransfected and vector transfected (pcDNA3) COS-7 cells did not express detectable levels of either the amidase or synthase. However, when COS-7 cells were transiently transfected with a rat FAAH pcDNA3 construct, both amidase and synthase were concomitantly expressed. These results indicate that the enzymatic formation of anandamide from arachidonic acid and ethanolamine can be mediated by anandamide amidase acting in the reverse direction. The FAAH transfected cells expressed higher levels of enzyme than either rat brain homogenates or neuroblastoma cells in culture. Furthermore, the reaction rate for the amidase in FAAH transfected COS-7 cells, neuroblastoma cells and brain homogenate was always greater than the synthase reaction. These studies raise the question if this synthase reaction serves any physiological role, especially in view of the evidence that anandamide can be formed by a different pathway.
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PMID:The cloned rat hydrolytic enzyme responsible for the breakdown of anandamide also catalyzes its formation via the condensation of arachidonic acid and ethanolamine. 934 46

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.
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PMID:Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae. 951 58

The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. We have examined the effect of VAI RNA on ADAR function. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation.
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PMID:Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase. 963 58

Cannabinoids are psychoactive components of marijuana, and bind to specific G protein-coupled receptors in the brain and other mammalian tissues. Anandamide (arachidonoylethanolamide) was discovered as an endogenous agonist for the cannabinoid receptors. Hydrolysis of anandamide to arachidonic acid and ethanolamine results in the loss of its biological activities. The enzyme responsible for this hydrolysis was solubilized, partially purified from the microsomes of porcine brain, and referred to as anandamide amidohydrolase. In addition to the anandamide hydrolysis, the enzyme preparation catalyzed anandamide synthesis by the condensation of arachidonic acid with ethanolamine. Several lines of enzymological evidence suggested that a single enzyme catalyzes both the hydrolysis and synthesis of anandamide. This reversibility was confirmed by the use of a recombinant enzyme of rat liver overexpressed in COS-7 cells. However, in consideration of the high Km value for ethanolamine as a substrate for the anandamide synthesis, the enzyme was presumed to act as a hydrolase rather than a synthase under physiological conditions. The recombinant enzyme acted not only as an amidase hydrolyzing anandamide and other fatty acid amides but also as an esterase hydrolyzing methyl ester of arachidonic acid. 2-Arachidonoylglycerol, which was found recently to be another endogenous ligand, was also efficiently hydrolyzed by the esterase activity of the same enzyme. The anandamide hydrolase and synthase activities were detected in a variety of rat organs, and liver showed by far the highest activities. A high anandamide hydrolase activity was also detected in small intestine but only after the homogenate was precipitated with acetone to remove endogenous lipids inhibiting the enzyme activity. The distribution of mRNA of the enzyme was in agreement with that of the enzyme activity.
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PMID:A hydrolase enzyme inactivating endogenous ligands for cannabinoid receptors. 986 62

Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabinoid receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine brain, and the cDNA encoded a protein of 579 amino acids with a molecular mass of 62.9 kDa. The amino acid sequence was 81, 80 and 85% identical with the enzymes previously cloned from the liver of rat, mouse, and human, respectively. When the enzyme protein was overexpressed in COS-7 cells, the particulate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidonic acid and ethanolamine both with a specific activity of 0. 2-0.3 micromol/min/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate specificity hydrolyzing oleamide, 2-arachidonoylglycerol, and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser-241, or Cys-249 completely abolished the hydrolyses of all the above-mentioned substrates as well as the synthesis of anandamide in the reverse reaction.
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PMID:Anandamide amidohydrolase of porcine brain: cDNA cloning, functional expression and site-directed mutagenesis(1). 1052 30


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