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)

Variants of the Cx43 gap junction protein have been detected on Western immunoblots by using an antipeptide antibody to the N-terminus of the protein. In heart ventricle, atrium, brain, retina, and uterus, different yet characteristic ratios of a broad 43-kDa band and a 39- to 40-kDa doublet were observed. These proteins (in lens epithelium, testes, and spleen) or their messages (in stomach, duodenum, kidney, and lung) were also detected in several nonexcitable systems but at consistently lower levels than found in electrically excitable tissues. The reproducible heterogeneity in electrophoretic mobility of Cx43 seen in different tissues does not appear to be due to proteolysis, since both the 43-kDa band and the 39- to 40-kDa doublet were recognized by an N-terminal as well as a C-terminal antibody. Furthermore, Northern (RNA) blots from different tissues show that both polypeptide profiles arise from indistinguishable transcripts. The conversion by alkaline phosphatase treatment of a predominantly 43-kDa profile (in heart) to a 39- to 40-kDa profile (characteristic of brain and protein translated in vitro from the RNA) suggests that the observed electrophoretic heterogeneity arises from tissue-wide differences in the phosphorylation state of Cx43.
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PMID:Tissue-specific distribution of differentially phosphorylated forms of Cx43. 184 23

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.
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PMID:Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. 184 42

Poly[15(IPGVG),(RGYSLG)], where RGYSLG is a protein kinase site, was synthesized. On raising the temperature of a 5 mg/ml solution, this polypeptide undergoes an inverse temperature transition at 18 degrees C in which it folds into a contracted state by optimizing intramolecular hydrophobic interactions. Averaging the data of five experiments, phosphorylation by means of a 3':5' cyclic AMP dependent protein kinase to the extent of one phosphate in 360 residues raises the temperature of the folding transition to 32 degrees C. The shift is completely reversed on dephosphorylation by alkaline phosphatase. Phosphorylation is hereby shown to be the most potent chemical perturbation known for shifting the temperature of an inverse temperature transition, which has been shown to be an efficient mechanism for achieving chemomechanical transduction (mechanochemical coupling).
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PMID:Phosphorylation and dephosphorylation modulation of an inverse temperature transition. 185 15

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

Incubation of intact isolated symbiosomes with [gamma-32P]ATP, followed by isolation of the peribacteroid membrane and polypeptide analysis, showed that a single major polypeptide at 26 kDa was labelled. Antibodies raised against nodulin 26 reacted with a similar sized polypeptide. Incubation of the symbiosomes with alkaline phosphatase removed the label from this polypeptide. Pre-incubation with ATP stimulated malate accumulation by isolated symbiosomes, but only slightly (10-30%). Pre-treatment of symbiosomes with alkaline phosphatase inhibited malate uptake substantially and this inhibition was completely relieved by addition of ATP. The ATP stimulation of malate uptake was not affected by ATPase inhibitors. It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.
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PMID:Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules. 195 59

High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution not only to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichia coli alkaline phosphatase polypeptide by linker insertion mutagenesis of the phoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues (Sowadski, J. M., Foster, B. A., and Wycoff, H. W. (1981) J. Mol. Biol. 150, 245-272). Mutant protein enzymatic function was generally tolerant of insertions in exterior loops, but was inactivated by insertion within alpha-helical or beta-strand structural elements. We further demonstrate that these tolerant surface loops can serve as vehicles for high level expression and stabilization of larger foreign peptide sequences, using a 15-residue analogue of dynorphin as an example. Insertion of the dynorphin "guest" peptide probably caused only a local structural perturbation of the alkaline phosphatase carrier since the hybrid protein retained enzymatic activity, was exported efficiently to the periplasmic space, and could be purified by anion-exchange chromatography using a protocol developed for alkaline phosphatase itself. The gust peptide was recovered from one of these fusion proteins intact and in high yield by protease digestion in vitro and was then purified by cation-exchange chromatography to near homogeneity in a single step.
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PMID:Introduction of guest peptides into Escherichia coli alkaline phosphatase. Excision and purification of a dynorphin analogue from an active chimeric protein. 196 50

Immunoblotted protein samples from several strains of Mycoplasma hominis and from one strain of Mycoplasma arginini each contain a polypeptide of a molecular mass of 95,000 to 105,000 Da which binds immunoglobulin nonimmunologically. Immunoblots from these organisms were probed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin, conjugated goat immunoglobulin G (IgG) Fab fragments, and conjugated goat IgG Fc fragments. The polypeptide bound the goat anti-rabbit molecules and the Fab fragments but not the Fc fragments. These reactions could be blocked with nonimmune unconjugated goat IgG and unconjugated human IgM. Controls probed with alkaline phosphatase alone did not stain. Binding of the conjugated preparations to whole mycoplasmal cells was dependent on concentrations of both conjugate and cells for the goat anti-rabbit preparation and for Fab. The mycoplasmal polypeptide may be a light-chain-specific reactant.
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PMID:Identification of a mycoplasmal protein which binds immunoglobulins nonimmunologically. 203 76

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
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PMID:Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C. 204 Jun 2

The transcription and translation of the tellurite-resistance (TeR) genes of the HII incompatibility group plasmid, pHH1508a, were studied. The nucleotide (nt) sequence of the TeR region was determined and two possible open reading frames, tehA and tehB, were identified. The direction of transcription and translation of these genes was confirmed through the preparation of lacZ and phoA (encoding alkaline phosphatase) fusions. The transcription start point was identified in the sequence using RNA primer extension. The tehA gene codes for a 36-kDa polypeptide which is highly hydrophobic. The TehA protein appears to be located in the inner membrane of the bacterial cell since tehA fusions with both phoA and lacZ were obtained and expressed. The tehB gene codes for a 23-kDa polypeptide which appears to be relatively hydrophilic and is probably located in the cytoplasm. Both proteins were overproduced using a T7 RNA polymerase/promoter system. No nt or amino acid sequence homology could be found between this TeR determinant and the TeR genes from the IncHI-2 plasmid, pMER610, and the IncP alpha plasmid, RK2.
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PMID:Nucleotide sequence and overexpression of the tellurite-resistance determinant from the IncHII plasmid pHH1508a. 206 Jul 88

Abnormalities of tubular membrane structure and composition have been proposed as the primary defect in nephronophthisis (NEF). In order to characterize the protein composition of tubular cells in NEF, in vitro methods were developed to culture and propagate tubular cells obtained from biopsy fragments. Accordingly, microdissected cortical slices (1 x 3 mm) were first digested with collagenase and DNAse and then grown in RPMI medium supplemented with 10% NU serum and conditioned serum deriving from 3T3 cultures. At confluence, cultured cells from NEF showed characteristics which were typical of normal tubules, i.e. presence of cytokeratin and positivity for succinic dehydrogenase and alkaline phosphatase stainings, and presented no morphological alterations compared to cultured cells from normal tubular epithelium. Moreover, no difference was observed for fibronectin, collagen IV and laminin stains. Analysis by two-dimensional electrophoresis of cellular extracts revealed several changes in protein composition of NEF, the main one being the decrease in NEF cells of a polypeptide with a molecular weight of 120 kD and a pI of 4.8; this polypeptide was a constant finding in normal kidneys. These observations demonstrated that human tubular epithelial cells can be successfully cultured from very small biopsy fragments, which represents a new approach to the study of molecular disorders involving tubular cells in inherited disease. Cultured cells from NEF maintain the same morphological, immunological and cytochemical characteristics as normal tubular cells, but present a few alterations in polypeptide composition which may have pathogenetic relevance. A more careful analysis of these alterations is needed to define the molecular disorder(s) involving the tubule in NEF.
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PMID:Tubular epithelium culture from nephronophthisis-affected kidneys: a new approach to molecular disorders of tubular cells. 207 4


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