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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase. The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm. One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined. The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20. The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins.
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PMID:Use of gene fusions to determine a partial signal sequence of alkaline phosphatase. 11 91

Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides.
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PMID:Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes. 34 83

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.
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PMID:In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. 136 26

Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [35S]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.
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PMID:Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. 184 32

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence. 191 27

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.
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PMID:Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase. 193 59

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.
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PMID:The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. 219 39

All batches of Met-hGH examined stimulated statural growth to approximately the same extent. The growth rates measured partly exceeded the results obtained in previous studies with pituitary preparations in the same dosage. Under treatment with SI, i.e. the preparation with the highest amount of ECP, high antibody titres with high binding capacity against GH and ECP were found. With SII all antibody determinations showed much lower titres. With Somatonorm (SIII), in the large majority of cases no antibodies were detectable. The titres registered in a few children were low and the binding capacities were negligible. The biologically determined somatomedin activity was initially pathologically low. During treatment it rose to supraphysiological levels. Also the radioimmunologically assayed somatomedin and the alkaline phosphatase increased significantly. At the start of the first series, two patients showed allergic skin reactions which turned out to be caused by the insufficiently purified preparations. Therapy with extractive preparations was free of such side-effects and fully successful. Both of the patients were atopic. A third child who was also allergic developed after 6-9 months the highest antibody titres seen, combined with a high binding capacity. Also, with this boy, treatment was switched over to pit-hGH, with very good results. Two children with pituitary dwarfism already developed in utero high antibody titres against Met-hGH but not against ECP. For this response, neither the Somatonorm nor its impurities can be implicated. Rather, it is the reaction to GH generally, which the organism recognizes as a foreign protein and thus as an antigen. One of the patients stopped growing after nine months. Likewise, pituitary GH did not lead to any further improvement.
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PMID:Recombinant human growth hormone. 242 93

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.
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PMID:Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils. 251 98

The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.
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PMID:Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase. 283 85


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