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)

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.
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PMID:Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue. 820 80

Neuronal protein synthesis is severely depressed following stress such as heat-shock, hypoxia, and hypoglycemia. Following reversible cerebral ischemia, protein synthesis is transiently inhibited in ischemia-resistant areas, but persistently depressed in vulnerable brain regions. Eukaryotic initiation factor 2 (eIF-2) activity, that is, the formation of the ternary complex eIF-2.GTP.initiator 35S-Met-tRNA, a rate-limiting step in the initiation of cellular protein synthesis, was studied in the rat brain during and following 15 min of transient global cerebral ischemia. At 30 min and 1 hr of reperfusion, a general decrease of eIF-2 activity by approximately 50% was seen in the postmitochondrial supernatant (PMS). In the relatively resistant neocortex and CA3 region of the hippocampus, the eIF-2 activity returns to control levels at 6 hr of reperfusion, but remains depressed in the vulnerable striatum and the CA1 region. Similarly, the activity of the guanine nucleotide exchange factor (GEF), which catalyzes the exchange of GTP for GDP bound to eIF-2, a crucial step for the continued formation of the ternary complex, is transiently reduced in neocortex but persistently depressed in striatum. The postischemic decrease in eIF-2 activity is further attenuated by agarose-bound alkaline phosphatase, and mixing experiments revealed that a vanadate-sensitive phosphatase may be responsible for the depression. Addition of partially purified GEF to PMS from postischemic neocortex restored eIF-2 activity to control levels. We conclude that ischemia alters the balance between phosphorylation and dephosphorylation reactions, leading to an inhibition of GEF and a depression of ternary complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stress-induced inhibition of protein synthesis initiation: modulation of initiation factor 2 and guanine nucleotide exchange factor activities following transient cerebral ischemia in the rat. 847 77

The factors responsible for predisposition to progressive organ injury and vascular complications in arterial hypertension are uncertain. Recent evidence shows that leukocytes participate in cardiovascular conditions for which hypertension is a risk factor. Therefore, there is a need to define the properties of circulating leukocytes in hypertensives. There are about twice as many circulating leukocytes in spontaneous hypertensive rats (SHRs) compared with their normotensive controls, the Wistar-Kyoto rats (WKYs). The SHR neutrophils are viscoelastic and similar to neutrophils in WKYs but exhibit lower deformability in short-term elastic deformation. Mature SHRs have elevated levels of spontaneous pseudopod formation. Mild stimulation with N-formyl-Met-Leu-Phe or platelet-activating factor (10(-8) M) results in a significantly enhanced level of neutrophil pseudopod formation in SHRs but not in WKYs. SHRs exhibit higher levels of spontaneous superoxide formation. Alkaline phosphatase content of individual circulating neutrophils in SHRs is on average lower while plasma levels of alkaline phosphatase in the same samples are elevated in the SHRs. Spontaneous degranulation of SHR neutrophils is also detectable with myeloperoxidase measurements. Such activity of circulating leukocytes poses a significant risk for vascular cytotoxicity in the hypertensive rats.
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PMID:Properties of circulating leukocytes in spontaneously hypertensive rats. 870 19

An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.
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PMID:An enzyme-linked immunosorbent assay for detecting proteolytic activity of hepatitis C virus proteinase. 917 84

In very recent studies it was established that transforming growth factor beta (TGF-beta), likely to be the most relevant fibrogenic cytokine and regulator of cell proliferation, differentiation, and matrix metabolism, is expressed by hepatocytes (parenchymal cell [PC]) and secreted from cultured PC in a latent form incapable of receptor binding. The structural composition of the latent TGF-beta complex secreted by cultured PC is unknown. In some TGF-beta expressing cell types this cytokine is released as a large molecular weight complex containing in addition to the TGF-beta latency associated peptide (LAP) a disulfide bonded latent TGF-beta binding protein (LTBP), of which the existence and function in liver is hitherto unknown. This study is directed to the identification of LTBP expression in rat PC. Cells were isolated from rat liver with the collagenase method and analyzed for LTBP before and during culture under standard conditions using alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostainings, metabolic labeling, messenger RNA (mRNA) detection (reverse-transcription polymerase chain reaction [RT-PCR]) and sequencing, and immunoblotting of gel chromatographically separated cell extracts and conditioned media, respectively. APAAP immunostainings applying a specific polyclonal LTBP-antiserum (ab 39) indicated expression of LTBP in PC of liver in situ and freshly isolated PC but a strong expression in cultured PC. Transcripts of LTBP-1 were detected by RT-PCR and confirmed by sequence analyses. Metabolic labeling of PC with [35S]-Met/Cys followed by immunoprecipitation of cell lysates with LTBP antiserum confirmed the synthesis of the high molecular mass complex of 250 kd containing LTBP with a molecular mass of 160 kd. Latent TGF-beta complexes, associated with LTBP related proteins, could be separated from both extracts and conditioned media of PC by gel filtration chromatography. They confirmed the release of the large latent TGF-beta complex from PC. Investigations of immunocytochemical LTBP staining under different culture conditions (TGF-beta supplementation, extracellular matrices) point to concordant variations of LTBP and TGF-beta expression. The results suggest a role for PC in paracrine- and autocrine-mediated effects of TGF-beta, which might be important for various cell activities in healthy and diseased liver.
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PMID:Expression and release of the latent transforming growth factor beta binding protein by hepatocytes from rat liver. 918 59

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.
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PMID:Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. 1079 69

Schistosoma mansoni egg antigens are mostly responsible for the granulomatous pathology in human intestinal schistosomiasis. Several previous studies have indicated that the induction of an immune response against some parasite enzymes may protect against pathology. The present work was designed to identify enzyme activities present in a standard soluble egg antigen (SEA) preparation. Simple colorimetric analyses were performed incubating SEA with 2-naphthyl, 2-naphthylamide (2NA), or p-nitrophenyl substrates at different pHs in the absence of added effectors. Results showed prominent acid phosphatase (pH 5.4), alkaline phosphatase (pH 8.5), and N-acetyl-beta-glucosaminidase (pH 5.4) activities. Relevant peptidase activities were also detected at pH 6.5-7.5 against 2NA derivatives of (1) aliphatic (alpha-Ala > beta-Ala > Leu > Met > S-benzyl-Cys), polar (Ser > Gln), basic (Arg > Lys > ornithine), and acidic (Glu) amino acids; (2) dipeptides: X-Ala (X = Gly > Leu > Lys > Asp), X-Arg (X = Ala > Arg > Phe > Gly > Pro > Asp), Ser-Met, and Phe-Pro; and (3) tripeptides (Ala-Phe-Pro > Phe-Pro-Ala). The data demonstrated that S. mansoni SEA contains a rich set of hydrolases with different specificities that might play a role in the egg physiology and possibly also in the host-parasite relationships.
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PMID:Enzyme activities in Schistosoma mansoni soluble egg antigen. 1112 95

Atlantic cod is a marine fish that lives at low temperatures of 0-10 degrees C and contains a cold-adapted alkaline phosphatase (AP). Preparations of AP from either the lower part of the intestines or the pyloric caeca area were subjected to proteolytic digestion, mass spectrometry and amino acid sequencing by Edman degradation. The primary structure exhibits greatest similarity to human tissue non-specific AP (80%), and approximately 30% similarity to AP from Escherichia coli. The key residues required for catalysis are conserved in the cod AP, except for the third metal binding site, where cod AP has the same variable residues as mammalian APs (His153 and His328 by E. coli AP numbering). General comparison of the amino acid composition with mammalian APs showed that cod AP contains fewer Cys, Leu, Met and Ser, but proportionally more Asn, Asp, Ile, Lys, Trp and Tyr residues. Three N-linked glycosylation sites were found. The glycan structure was determined as complex biantennary in type with fucose and sialic acid attached, although a trace of complex tri-antennary structure was also observed. A three-dimensional model was obtained by homology modelling using the human placental AP scaffold. Cod AP has fewer charged and hydrophobic residues, but more polar residues at the intersubunit surface. The N-terminal helix arm that embraces the second subunit in dimeric APs may be more flexible due to a replaced Pro at its base. One disulfide bridge was found instead of the two present in most other APs. This may invoke greater movement in the structure that together with weaker subunit contacts leads to improved catalytic efficiency.
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PMID:Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua). 1294 38

Neuromuscular diseases are a known risk factor for immobilization-induced osteoporosis. The aim of the study was to analyse bone mineral density (BMD) in patients with familial amyloid polyneuropathy (FAP) type I (Val30 Met) and to compare them with a population of patients with other neuromuscular disorders. We studied 24, ambulatory, neuromuscular patients, all men and premenopausal women. We included 12 FAP patients (GI) and 12 patients with other disorders (GII). Clinical data included age, sex, height, weight, alcohol intake, smoking, calcium intake, physical activity and history of fractures. Serum and urinary calcium, osteocalcin, bone alkaline phosphatase, parathyroid hormone, thyroid stimulating hormone and urinary N-telopeptide cross-linked type 1 collagen were determined in all patients. Bone mineral density of lumbar spine, hip and wrist were determined by dual energy X-ray absorptiometry scan. No statistical differences were found in clinical or analytic data between the two groups, except for body mass index and calciuria, which were lower in GI. In GI, 54.5% were osteoporotic, against 23.1% in GII (P = 0.04). Bone mineral density was lower in GI when compared with GII, and tended to decrease with disease duration. Decreased BMI and the early autonomic involvement in GI probably explain the results. The prevention and early treatment of osteoporosis, in FAP patients should be considered a priority.
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PMID:Bone mineral density in familial amyloid polyneuropathy and in other neuromuscular disorders. 1588 54

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MS(n) (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.
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PMID:Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family. 1627 Oct 39


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