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)

Human erythrocytes from healthy male donors were fractionated with respect to in vivo age by simple centrifugation in order to characterize changes in the functional integrity of the membrane during the life-span of the cell. The three enzymes, Na/K-ATPase, glyceraldehyde-3-phosphate dehydrogenase and NADH-ferricyanide reductase, were found not to change with age, but significant age-dependent decreases were observed in the cases of acetylcholinesterase, phosphoglycerate kinase, purine nucleoside phosphorylase, adenylate kinase, Mg-ATPase and alkaline phosphatase. The possibility that these changes were attributable to mechanisms other than age-related inactivation, such as reticulocyte contamination, differential resealing and crypticity, was investigated. Only the decrease in acetylcholinesterase could be explained wholly in terms of reticulocyte contamination. A decrease in membrane integrity on ageing was observed, which accounted for approximately half the change in alkaline phosphatase and may have contributed to the other enzyme activity changes. This membrane integrity effect masked a real decrease in the highly cryptic NADH-ferricyanide reductase, this decrease being apparent only after total disaggregation of the membrane with nonionic surfactant.
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PMID:Changes in the activities of some membrane-associated enzymes during in vivo ageing of the normal human erythrocyte. 14 40

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
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PMID:Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase. 22 19

In order to investigate the possible involvement of corticotropin-releasing factor (CRF) and somatostatin (SRIF) on thyrotropin-releasing hormone (TRH) neuronal cell activity in the rat hypothalamic paraventricular nucleus, we have proceeded to the simultaneous localization of CRF or SRIF and TRH. For this purpose, we used a dual immunostaining procedure that employed antibodies to CRF and SRIF and peroxidase-labeled goat anti-rabbit IgG as a first sequence, and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive CRF and SRIF fibers was observed. A large number of CRF and SRIF nerve endings were seen intimate anatomic proximity and often appeared to surround TRH-containing cell bodies. These results strongly suggest that TRH neurons might be regulated by both CRF and SRIF. These interactions might be the neuroanatomical basis for the already observed inhibitory effects of CRF and SRIF on TRH release.
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PMID:Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus. 135 8

This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.
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PMID:Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae. 139 67

This study evaluates five cryptic plasmid-derived DNA probes in a 4-h slot-blot hybridization assay for the detection of Chlamydia trachomatis in cultures and clinical specimens. The probes, consisting of either the entire cloned 7.5 kbp cryptic plasmid pSE8 or one of four Hin dIII/Eco RI fragments measuring 710, 1055, 710, and 500 bp, respectively, were labelled with Photoprobe biotin. The probe was detected using a streptavidin-alkaline phosphatase conjugate followed by addition of BCIP and NBT. The sensitivity of the assay, using 25 ng of probe DNA, ranged from 50 pg (with the entire plasmid as probe) to 5 ng (with the 500 bp fragment as probe). A total of 103 reference strains of Chlamydia trachomatis and other bacteria were tested for reactivity with the probes. All 18 reference strains of C. trachomatis hybridized with the probes. None of the DNA from the heterologous organisms tested was found to hybridize with any of the probes. A total of 174 samples taken from patients visiting the STD clinic at the University Hospital, University of Seville were used in the study. The overall sensitivity of the assay, using the 710 bp biotinylated probe was 94.5% compared to culture while the specificity was 97.5%. Positive and negative predictive values were 96.5% and 97.5%, respectively. It appears that the plasmid-derived probes used in this study could serve as useful tools for the rapid and specific detection of Chlamydia trachomatis in cell cultures and clinical specimens.
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PMID:Evaluation of a DNA probe of plasmid origin for the detection of Chlamydia trachomatis in cultures and clinical specimens. 177 80

In order to determine the nature of afferent fibres contacting thyrotropin-releasing hormone (TRH)-synthesizing neuronal cell bodies in the rat hypothalamic paraventricular nucleus, we used dual immunostaining procedures which employed antibodies to ACTH (to label proopiomelanocortin (POMC) neurons), neuropeptide Y (NPY) and dopamine-beta-hydroxylase (D beta H) and peroxidase-labeled goat anti-rabbit IgG as a first sequence and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive POMC, NPY and D beta H fibres was observed. Numerous NPY and POMC fibres were in intimate anatomic proximity and often appeared to surround in remarkable density TRH-containing cell bodies. Less frequent appositions between D beta H fibres and TRH cell bodies were detected. These results strongly suggest that TRH neurons might be regulated by POMC, NPY as well as adrenergic and/or noradrenergic systems. These interactions might be the neuroanatomical basis for the already observed effects of opiate peptides, NPY and catecholamines on TSH secretion.
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PMID:Anatomical interactions of proopiomelanocortin (POMC)-related peptides, neuropeptide Y (NPY) and dopamine beta-hydroxylase (D beta H) fibers and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus. 190 63

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.
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PMID:Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations. 193 29

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

Mutants of Escherichia coli have been selected for the absence of 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (2',3'-cyclic phophodiesterase). Mutants selected for the absence of 5'-nucleotidase are of two kinds: those that lack detectable activity for the enzyme (Ush(-)), and those that possess activity when cell extracts are assayed, but not when intact cells are assayed (cryptic; Crp(-)). The latter class is probably identical to a type of mutant previously reported by Ward and Glaser. When mutants are selected for the absence of 3'-nucleotidase, Crp(-)mutants are also obtained. Thus far, however, mutants totally lacking this enzyme have not been found. The location on the genetic map of one ush mutation is at position 11 min and that of one crp mutation at approximately 67 min. In the crp mutant, 5'-nucleotidase and 3'-nucleotidase remain located in the periplasm. This mutant is also cryptic for alkaline phosphatase but not for acid hexose phosphatase. Treatment of cells with ethylenediamine-tetraacetate substantially alleviated crypticity. These data are discussed in terms of the organization of periplasmic enzymes and of the outer membrane as a permeability barrier.
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PMID:Mutants of Escherichia coli K-12 "cryptic," or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (cyclic phosphodiesterase) activity. 435 92

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.
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PMID:Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis. 776 42


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