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)

A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.
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PMID:Inhibition of Bacillus subtilis growth and sporulation by threonine. 10 59

Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.
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PMID:A receptor binding domain of mouse interleukin-4 defined by a solid-phase binding assay and in vitro mutagenesis. 160 64

Experiments were carried out to study the digestibility of a cassava (gari) diet and its effect on growth in young male dogs. Three groups of dogs were fed on diets with rice (control), cassava (gari), and rice + cyanide respectively as the carbohydrate source. Each diet contained 130 g crude protein (nitrogen x 6.25)/kg, was supplemented with vitamins and minerals, and was fed for 14 weeks. Variables measured were body-weight gain, bone growth, plasma alkaline phosphatase (EC 3.1.3.1) activity, total serum 3,5,3'-triiodothyronine (T3) and some plasma free amino acids. The apparent digestibilities of dry matter, protein and fat were not significantly different in the three groups, but the digestibility of gari fibre was significantly lower than the digestibility of rice fibre when fed to dogs (P less than 0.05). Proximate analysis of the faeces showed that the group of dogs fed on the gari diet had faeces which had a significantly higher moisture content than the faeces of the other groups (P less than 0.05), and also a significantly higher fibre content (P less than 0.05). There was no significant difference in body-weight gain and bone growth between the control and gari-fed groups of dogs, but these variables were significantly lower in the dogs fed on the rice + cyanide diet (P less than 0.05). At the end of the 14-week experimental period total serum T3 and plasma alkaline phosphatase activity were not significantly different between the control group of dogs and the gari-fed group, but were significantly lower in the rice + cyanide group. Plasma free methionine, leucine, isoleucine and valine concentrations were higher in the rice + cyanide group of dogs than in the control group and the gari group, indicating that these amino acids were accumulating and not being utilized for protein synthesis and growth to the same extent in the rice + cyanide group of dogs as in the other groups. It was concluded that the digestibilities of cassava starch and rice starch were the same in the dog but that rice fibre was more digestible in the dog than cassava fibre. It was also concluded that growth proceeded normally when a balanced gari diet or a balanced rice diet containing 130 g crude protein/kg was fed to dogs, but growth was retarded when a balanced rice + cyanide diet containing 130 g crude protein/kg was fed to dogs because total serum T3 concentration became greatly depressed.
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PMID:Digestibility of a nutritionally-balanced cassava (Manihot esculenta Crantz) diet and its effect on growth in young male dogs. 166 68

p19 is a highly conserved 19-kDa cytosolic protein that undergoes phosphorylation in mammalian cells upon activation of several distinct signal transduction pathways. Its expression is widespread but developmentally regulated. To determine the in vivo phosphorylation site(s) of p19, the protein was purified from bovine brain and resolved into the unphosphorylated form (p19) and a mixture of the two predominant phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses corresponding to fragments spanning the entire amino acid sequence as deduced from the cDNA except for those predicted to contain an unmodified amino terminus. Instead, the digests revealed ions corresponding to peptides lacking the initiator methionine and containing an N-acetylated alanine at the amino terminus. The analysis of pp19, but not that of p19, revealed two sets of ions representing peptides whose m/z values differed by 80 atomic mass units, the incremental mass of a phosphate residue. These putative phosphate-bearing peptides were sensitive to alkaline phosphatase treatment. Using combined trypsin and V8 protease digestions, the phosphorylation sites were mapped to Ser-25 and Ser-38, in the peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys, respectively. Interestingly, both phosphoserines are in a very similar sequence context, suggesting that a single proline-directed serine protein kinase, possibly p34cdc2, is responsible for phosphorylation of both sites in vivo.
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PMID:Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry. Identification of two proline-directed serine phosphorylation sites and a blocked amino terminus. 173 1

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

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.
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PMID:A lipopeptide-encoding sequence upstream from the lysA gene of Pseudomonas aeruginosa. 211 74

We have examined the hydrophobicity component of signal peptide function using polymeric sequences in combination with cassette mutagenesis. Using homopolymeric units of either isoleucine, leucine, valine or alanine to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for export and processing were delineated. The transport properties of these mutants demonstrated that the net hydrophobicity determines the total extent of precursor processing, while a high mean hydrophobicity/residue is critical for complete, rapid processing and translocation. Moreover, alkaline phosphatase was converted from a periplasmic to an active membrane-anchored protein via a signal containing 20 leucine residues. This application of polymeric sequences allows systematic comparisons to be made, unambiguously revealing the hydrophobicity requirements governing specific steps in the transport process.
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PMID:Polymeric sequences reveal a functional interrelationship between hydrophobicity and length of signal peptides. 215 63

SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments. Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo. Both the SecY and PhoA products of this cleavage have been identified immunologically. In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase. The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase. A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells. These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally. Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.
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PMID:SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site. 218 44

The effect of lithium and other antipsychotic drugs on the renal function in patients with manic-depressive disorders has been investigated. Thirty-four patients (5 males and 29 females) treated with lithium and 21 patients (6 males and 15 females) on other antipsychotic drugs were studied. A control group of 10 persons consisting of healthy subjects, all of whom were taking no medication was also studied. No significant differences in the treatment duration were present between the patients investigated. Although few patients on lithium had glomerular filtration reduced, no statistically significant difference in creatinine clearance was found between the groups. None of the patients had a disturbance in the reabsorption of glucose, amino acids (histidine, lysine, valine, glutamine, glycine, serine, taurine, threonine, alanine, isoleucine) and beta 2-microglobulin. Patients treated with lithium had a significantly reduced urine concentration and higher daily diuresis than did the other two studied groups. A significantly higher overnight elimination of alkaline phosphatase was found in a group of patients taking other antipsychotic drugs. The attained results suggest tubular lesions in patients with manic-depressive psychosis occurring in the association with the prophylactic use of lithium and, at same time, the possibility of the other in association with the other antipsychotic drugs.
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PMID:[Effect of long-term use of lithium on kidney function]. 236 20


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