The catalytic-centre activity and kinetic properties of bovine milk alkaline phosphatase.
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ABSTRACT:
SUBMITTER: Barman TE
PROVIDER: S-EPMC1270129 | biostudies-other | 1966 Nov
REPOSITORIES: biostudies-other
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Project description:The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.
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Project description:BACKGROUND:Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth. METHODS:Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40?weeks and in 289 breast milk samples collected from 78 individual mothers between days 2-49 post-birth. RESULTS:We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2-7. CONCLUSIONS:Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.
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Project description:The FEEDMI Study (NCT03663556) evaluated the influence of infant feeding (mother's own milk (MOM), donor human milk (DHM) and formula) on the fecal microbiota composition and alkaline phosphatase (ALP) activity in extremely and very preterm infants (≤32 gestational weeks). In this observational study, preterm infants were recruited within the first 24 h after birth. Meconium and fecal samples were collected at four time points (between the 2nd and the 26th postnatal days. Fecal microbiota was analyzed by RT-PCR and by 16S rRNA sequencing. Fecal ALP activity, a proposed specific biomarker of necrotizing enterocolitis (NEC), was evaluated by spectrophotometry at the 26th postnatal day. A total of 389 fecal samples were analyzed from 117 very preterm neonates. Human milk was positively associated with beneficial bacteria, such as Bifidobacterium, Bacteroides ovatus, and Akkermancia muciniphila, as well as bacterial richness. Neonates fed with human milk during the first week of life had increased Bifidobacterium content and fecal ALP activity on the 26th postnatal day. These findings point out the importance of MOM and DHM in the establishment of fecal microbiota on neonates prematurely delivered. Moreover, these results suggest an ALP pathway by which human milk may protect against NEC.
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Project description:In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein functional evolution.
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Project description:Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn(2+)-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase activity). The best deoxyribozyme decreases the half-life for phosphoserine hydrolysis from as high as >10(10) y to <1 h. The phosphatase activity also occurs with nonpeptidic substrates but with reduced efficiency, indicating a preference for phosphopeptides. The newly identified deoxyribozymes can function with multiple turnover using free peptide substrates, have activity in the presence of human cell lysate or BSA, and catalyze dephosphorylation of a larger protein substrate, suggesting broader application of DNA catalysts as artificial phosphatases.
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Project description:Aromatic residues of bovine kidney alkaline phosphatase appear to be involved in the interaction with ascorbate, as shown by the strong quenching of intrinsic fluorescence and absorption. Difference u.v.-absorption spectra clearly indicate that conformational changes also occur. The pH value at which the greatest fluorescence deactivation is found is close to that necessary for optimal catalytic activity and for maximal inhibition by ascorbate. A protective effect against ascorbate is afforded by Pi. Time profiles of inactivation on one side and of absorbance and emission quenching on the other display opposite behaviours. Attempts to reverse the effects by the use of KOH fail to restore enzyme activity or to modify the spectral effects of ascorbate. The protein alterations are related, directly or indirectly, to the enzyme active centre and can be probably ascribed to the redox and chelating properties of ascorbate.
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Project description:Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.
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Project description:Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.
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