Differential regulation of mRNAs encoding for rat intestinal alkaline phosphatase

1990 ◽  
Vol 259 (1) ◽  
pp. G93-G98 ◽  
Author(s):  
R. Eliakim ◽  
S. Seetharam ◽  
C. C. Tietze ◽  
D. H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Cdna Probe ◽  
Fold Increase ◽  
Maximal Activity ◽  
Amino Acid Residues ◽  
Intestinal Alkaline Phosphatase ◽  
Mrna Accumulation ◽  
Dihydroxyvitamin D3 ◽  
Fat Feeding

A cDNA probe encoding the entire structural region of the 62-kDa rat intestinal alkaline phosphatase from amino acid residues 1 to 531 detected multiple mRNA species (3.0, 2.7, and 2.2 kb) in rat intestinal RNA. The 3.0-kb species was most evident in duodenum but could be easily detected in jejunum using a 48-mer oligonucleotide encoding amino acid residues 492-508. This 48-mer oligonucleotide bound preferentially to the 3.0-kb mRNA, suggesting that the 2.7-kb mRNA differed in this region. To determine whether each of the mRNAs encoding rat intestinal alkaline phosphatase responded coordinately to physiological stimuli, the full-length cDNA and the 48-mer oligonucleotide were used as probes for the 2.7- and 2.2-kb and the 3.0-kb mRNAs, respectively. Intestinal mRNA concentration was measured by Northern blot analysis in acute (single feed, 17 kcal) and chronic (3 wk, 30% fat diet) fat feeding and in rachitic rats after 1,25-dihydroxyvitamin D3 therapy. There was a large increase (8- to 25-fold) in the 3.0-kb mRNA 7 h after acute fat feeding, with a much smaller increase (1.4- to 5.0-fold) in the 2.7- and 2.2-kb species. The peak in 3.0-kb mRNA accumulation correlated in time with the maximal activity of serum phosphatase activity after acute fat feeding (4- to 5-fold increase). In contrast, there was a much smaller increase in all mRNAs and in tissue and serum enzyme activity after chronic fat feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Primary structure of rat liver alkaline phosphatase deduced from its cDNA

1988 ◽  
Vol 249 (3) ◽  
pp. 661-668 ◽  
Author(s):  
Y Misumi ◽  
K Tashiro ◽  
M Hattori ◽  
Y Sakaki ◽  
Y Ikehara
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Rat Liver ◽  
Blot Hybridization ◽  
Fold Increase ◽  
Single Species ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Duct Ligation

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.

scholarly journals Role of methionine in the active site of α-galactosidase from Trichoderma reesei

1995 ◽  
Vol 308 (3) ◽  
pp. 955-964 ◽  
Author(s):  
A M Kachurin ◽  
A M Golubev ◽  
M M Geisow ◽  
O S Veselkina ◽  
L S Isaeva-Ivanova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Trichoderma Reesei ◽  
Active Site ◽  
Fold Increase ◽  
Thiol Group ◽  
Competitive Inhibitor ◽  
Amino Acid Residues ◽  
Activity Curve ◽  
Oxidative Activation ◽  
Alpha Galactosidase

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

scholarly journals Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

2002 ◽  
Vol 68 (6) ◽  
pp. 2676-2682 ◽  
Author(s):  
Daohai Zhang ◽  
Xianzhen Li ◽  
Lian-Hui Zhang
Keyword(s):  
Amino Acid ◽  
Sequence Alignment ◽  
Half Life ◽  
Bacterial Isolate ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Maximum Activity ◽  
Amino Acid Residues ◽  
Sequence Domain ◽  
Gene Cloning And Characterization

ABSTRACT The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)8-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp241, Glu295, Asp369, His145, and His368) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu498 and Arg310 with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

1999 ◽  
Vol 65 (7) ◽  
pp. 3001-3007 ◽  
Author(s):  
Frederic Chavagnat ◽  
Michael G. Casey ◽  
Jacques Meyer
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Streptococcus Thermophilus ◽  
Maximal Activity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
T7 Promoter ◽  
Reading Frame ◽  
Endopeptidase Activity

ABSTRACT The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.

The Two mRNAS Encoding Rat Intestinal Alkaline Phosphatase Represent Two Distinct Nucleotide Sequences

1992 ◽  
Vol 38 (12) ◽  
pp. 2506-2509 ◽  
Author(s):  
M J Engle ◽  
D H Alpers
Keyword(s):  
Polymerase Chain Reaction ◽  
Alkaline Phosphatase ◽  
Duodenal Mucosa ◽  
Differential Regulation ◽  
Untranslated Regions ◽  
Terminal Sequence ◽  
Intestinal Alkaline Phosphatase ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Fat Feeding

Abstract Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.

Rapid Communication: Ileal alkaline phosphatase is upregulated following functional amino acid supplementation in Salmonella Typhimurium-challenged pigs

2021 ◽  
Author(s):  
Lucas A Rodrigues ◽  
Michael O Wellington ◽  
J Caroline González-Vega ◽  
John K Htoo ◽  
Andrew G Van Kessel ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Salmonella Typhimurium ◽  
Experimental Period ◽  
Post Inoculation ◽  
Adaptation Period ◽  
Intestinal Alkaline Phosphatase ◽  
Weanling Pigs ◽  
Plasma Alkaline Phosphatase ◽  
Dietary Treatments

Abstract We recently showed that functional amino acid (FAA) supplementation improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs which was further improved by a longer adaptation period. It is expected that the effects are associated with increased activity of intestinal alkaline phosphatase (IAP). The objective of this study was to evaluate the effects of FAA supplementation and adaptation period on the ileal, cecal, and colonic activity of IAP in weaned pigs challenged with ST. In Exp. 1, 32 mixed-sex weanling pigs were randomly assigned to dietary treatments in a 2 × 2 factorial arrangement with low (LP) or high protein (HP) content and basal (FAA–) or FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements) as factors. In Exp. 2, a total of 32 mixed-sex weanling pigs were randomly assigned to 1 of 4 dietary treatments, being FAA- fed throughout the experimental period (FAA−) or a FAA profile fed only in the post-inoculation (FAA+0), for 1 wk pre- and post-inoculation (FAA+1), or throughout the experimental period (FAA+2). In Exp. 1 and 2, after a 7- and 14-d adaptation period, respectively, pigs were inoculated with saline solution containing ST (3.3 and 2.2 × 10 9 CFU/mL, respectively). Plasma alkaline phosphatase was measured on d 0 and 7 post-inoculation in Exp. 1, and IAP (ileum, cecum, and colon) was measured in Exp. 1 and 2. Correlations among ileal IAP and serum albumin and haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and reduced:oxidized glutathione (GSH:GSSG), ileal myeloperoxidase (MPO), ST shedding and ileal colonization, and post-inoculation average daily gain (ADG), feed intake (ADFI), and gain:feed (G:F) were also analyzed. In Exp. 1, plasma alkaline phosphatase was decreased with ST inoculation and overall content was increased in LP-FAA+ compared to LP-FAA- (P < 0.05). Moreover, ileal IAP was increased in FAA+ compared to FAA- pigs in both studies (P < 0.05) regardless of adaptation time (P > 0.05). Intestinal alkaline phosphatase was positively correlated with MDA and ADFI and negatively correlated with SOD and ST shedding in Exp. 1 (P < 0.05). These results demonstrate a positive effect of FAA supplementation, but not adaptation period, on ileal alkaline phosphatase activity in Salmonella-challenged pigs, which may be associated with improvements in antioxidant balance.

scholarly journals The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation

2002 ◽  
Vol 76 (2) ◽  
pp. 473-483 ◽  
Author(s):  
Tatineni Satyanarayana ◽  
Siddarame Gowda ◽  
María A. Ayllón ◽  
María R. Albiach-Martí ◽  
Shailaja Rabindran ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Citrus Tristeza Virus ◽  
Rna Binding ◽  
Fold Increase ◽  
Amino Acid Residues ◽  
Rna Accumulation ◽  
P23 Gene ◽  
Tristeza Virus ◽  
Citrus Tristeza

ABSTRACT Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3′ genes expressed via a nested set of nine or ten 3′-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3′ genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3′-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3′ genes.

A novel phosphatase upregulated inAkp3knockout mice

2007 ◽  
Vol 293 (5) ◽  
pp. G1068-G1077 ◽  
Author(s):  
Sonoko Narisawa ◽  
Marc F. Hoylaerts ◽  
Kutbuddin S. Doctor ◽  
Michiko N. Fukuda ◽  
David H. Alpers ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Posttranslational Modifications ◽  
Sequence Similarity ◽  
Catalytic Efficiency ◽  
Intestinal Alkaline Phosphatase ◽  
Catalytic Rate Constant ◽  
Fold Reduction ◽  
Enhanced Expression ◽  
Mouse Intestine ◽  
Fat Feeding

Reexamination of the Akp3−/−mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3−/−gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner ( gIAP) as opposed to duodenum-specific IAP ( dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant ( kcat) and a 30% drop in Km, leading to a 4-fold reduction kcat/ Kmcompared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3−/−mice. Developmentally, Akp3 expression is turned on during postnatal days 13–15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3−/−mice fed a high-fat diet.

scholarly journals A Novel Gene Involved in Regulating the Flagellar Gene Cascade in Proteus mirabilis

2006 ◽  
Vol 188 (22) ◽  
pp. 7830-7839 ◽  
Author(s):  
Lindsay G. Stevenson ◽  
Philip N. Rather
Keyword(s):  
Cell Differentiation ◽  
Amino Acid ◽  
Proteus Mirabilis ◽  
Fold Increase ◽  
Single Copy ◽  
Flagellar Gene ◽  
Mrna Accumulation ◽  
Swarmer Cell ◽  
Novel Gene ◽  
Expression Of Genes

ABSTRACT In this study, we identified a transposon insertion in a novel gene, designated disA, that restored swarming motility to a putrescine-deficient speA mutant of Proteus mirabilis. A null allele in disA also increased swarming in a wild-type background. The DisA gene product was homologous to amino acid decarboxylases, and its role in regulating swarming was investigated by examining the expression of genes in the flagellar cascade. In a disA mutant background, we observed a 1.4-fold increase in the expression of flhDC, which encodes FlhD2C2, the master regulator of the flagellar gene cascade. However, the expressions of class 2 (fliA, flgM) and class 3 (flaA) genes were at least 16-fold higher in the disA background during swarmer cell differentiation. Overexpression of DisA on a high-copy-number plasmid did not significantly decrease flhDC mRNA accumulation but resulted in a complete block in mRNA accumulation for both fliA and flaA. DisA overexpression also blocked swarmer cell differentiation. The disA gene was regulated during the swarming cycle, and a single-copy disA::lacZ fusion exhibited a threefold increase in expression in swarmer cells. Given that DisA was similar to amino acid decarboxylases, a panel of decarboxylated amino acids was tested for effects similar to DisA overexpression, and phenethylamine, the product of phenylalanine decarboxylation, was capable of inhibiting both swarming and the expression of class 2 and class 3 genes in the flagellar regulon. A DisA-dependent decarboxylated amino acid may inhibit the formation of active FlhD2C2 heterotetramers or inhibit FlhD2C2 binding to DNA.

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