Catalytic Activity Of Alkaline Phosphatase Research Articles

Resorcinol monophosphate ester as a new substrate for fluorometric detection of alkaline phosphatase activity in milk products

Alkaline phosphatase (ALP) is an indicator of sterilization efficacy of milk products and its activity monitoring is essential for ensuring food safety and controlling product quality. Here, a novel fluorometric method for the determination of ALP activity based on resorcinol monophosphate ester (RME) as a new substrate was proposed to verify pasteurization of milk products. RME was cheap and was simply and novelly synthesized by a two-step method using resorcinol (RC) as the raw material. The mechanism is, after dephosphorylation, RME was transformed to RC, which subsequently react with dopamine (DA) to form a strong fluorescent product, azamonardine. It has a fluorescence emission wavelength at 461 nm under the optimal excitation at 418 nm. The results show that under the optimum conditions, there is a good linear relationship between ΔF (the variation of fluorescence intensity) and ALP activity over 0–5000 mU/L. High sensitivity was achieved (detection limit of 17.34 mU/L) because of high quantum yield of the products and catalytic activity of ALP and the recovery rate is 96.8–106.1%. Compared with national and international standard methods, the detection procedures are simple, just adding RME and DA solution to milk with buffer solution. Our method was successfully applied for ALP activity detection in milk products. Moreover, the catalyst of ALP and the reaction of RME and dopamine are pH dependent, and thus the reaction time could be kept identical by terminating the reaction through adding acids. With high sensitivity, high selectivity, simple operation, and wide linear range, our assay will find more practical applications in food analysis.

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Alkaline phosphatase (ALP), a homo‐dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP‐based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single‐molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP‐based quantitative digital bioassays. This study aims quantitative analysis of single‐molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half‐active and fully‐active portions. Digital assays with serial buffer exchange uncovered single‐molecule Michaelis–Menten kinetics of ALP; half‐active molecules have halved values of the catalytic turnover rate, k cat, and the rate constant of productive binding, k on, of the fully active molecules. These findings suggest that half‐active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell‐free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.

Кореляційний аналіз показників гемостазу і функціонального стану печінки за гострого калькульозного холециститу

Introduction.Acute calculous cholecystitis (ACC) is characterized by the development of certain symptoms – from the local inflammatory process to significant functional disorders in the liver. The activity of the hemostasis system is directly related to the functional state of the liver, since hepatocytes are the main site for the synthesis of most proteins, in particular the proteins of the blood coagulation system.The aim of the study – to establish the relationship between the hemostasis system and the functional state of hepatocytes in patients with acute calculous cholecystitis (ACC), uncomplicated and complicated by obstructive jaundice (OJ) using a correlation analysis.Research methods.The subject of the study was plasma and serum of 47 patients with uncomplicated АСС (group I) and 32 patients with АСС with OJ (group II). For comparison, serum and blood plasma of 25 virtually healthy individuals, who were the control group, were examined. The parameters of the hemostasis system platelet count (prothrombin time (PT), activated partial thromboplastin time (APTT), total fibrinogen concentration (TFC), soluble fibrin-monomer complexes (SFMC), antithrombin III (ATIII) and Hageman-dependent fibrinolysis activity were determined (HDF). The determination of indicators of the liver function of the catalytic activity of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT) and cholinesterase (CHE) and the concentration of α1-antitrypsin (α1-AT),α2-macroglobulin (α2-MG) and C-reactive protein (CRP) was also made.Results and Discussion. The revealed correlations allow determining the degree of conjugacy of processes. The less the number of bonds is the weaker conjugacy of processes, the more flexible becomes the system that responds to the pathological change of the internal environment. The greatest number of correlation bonds was established in the control group of patients. In patients with ACC without complications, there was a tendency to decrease the number of bonds, where α1-AT correlated most withthe investigated parameters. The reduction of the amount and the change of the structure of correlation bonds were found in patients with ACC with OJ. A new correlation between the studied parameters in this group of patients has also been established.Conclusions.The established correlation bonds in uncomplicated ACC and complicated OJ indicate the tension of the adaptive mechanisms of the hemostasis system and the functional state of the liver when the inflammatory process complicates.

Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification

MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3’ end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3′-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3′-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones.

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.

A New Approach to the Enzymatic Determination of Magnesium, Calcium, and Barium

The effects of magnesium, calcium, and barium ions on the catalytic activity of alkaline phosphatase from the small intestine of the Greenland seal in the reaction of p-nitrophenyl phosphate hydrolysis in the presence of a number of complexones (ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, 1,2-cyclohexanediaminetetraacetic acid, and nitrilotrimethylenephosphonic acid) were studied. As a result, a new approach to the enzymatic determination of the above metals was developed. It consists in the use of the liberative effects of these metals on alkaline phosphatase preinhibited by nitrilotrimethylenephosphonic acid. Sensitive procedures for the enzymatic determination of magnesium, calcium, and barium ions were developed (c min = 14 ng/mL, 24 ng/mL, and 0.8 µg/mL, respectively).

Transforming growth factor β1 immobilized adsorptively on Ti6Al4V and collagen type I coated Ti6Al4V maintains its biological activity

Titanium and titanium alloys are often used for orthopedic and dental implants. Osseointegration of Ti6Al4V may be improved not only by precoating of the surface with extracellular matrix proteins like collagen type I but also by additional immobilization of growth factors. In the present study, transforming growth factor β1 (TGF- β1) which is known as an inducer of collagen synthesis was immobilized adsorptively on uncoated and collagen type I coated Ti6Al4V surfaces. TGF- β1 was found immobilized slightly faster to collagen type I coated than to uncoated Ti6Al4V and released slower from the collagen coated material. Immobilized TGF- β1 is biologically active for at least 3 weeks storage at 4°C. Sterilization by ethylene oxide inactivates immobilized TGF- β1. In osteoblasts cultured on implants with adsorptively immobilized TGF- β1, mRNA level and specific catalytic activity of alkaline phosphatase as well as accumulation of calcium and phosphate were found reduced, whereas procollagen α1(I) mRNA level and the rate of collagen synthesis were increased.

Biotin-Avidin Mediated Homogeneous Enzyme-Linked Binding Assays for Riboflavin and Riboflavin Binding Protein Using Alkaline Phosphatase as the Labelt

The feasibility of using alkaline phosphatase (ALP) as a generic label in a homogeneous type assay is examined. In the proposed ALP-based biotin-avidin mediated assay, the activity of the ALP-biotin conjugate is inhibited in the presence of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount proportional to the concentration of riboflavin binding protein (RBP) added. The ALP activity of the mixture is re-inhibited upon additions of free analyte riboflavin, to an extent proportional to the concentration of riboflavin added to the assay mixture. An avidin-riboflavin conjugate with a desired inhibitory effect is prepared by protecting the binding-site of avidin with 2-iminobiotin during the conjugation reaction at pH 6.5. The catalytic activity of ALP, conjugated with biotin, is inhibited to a significant degree (up to 86%) by the binding of the avidin-riboflavin conjugate, and is fully recovered upon additions of RBP.

Catalytic activities of alkaline phosphatase and N-acetyl-beta-D-glucosaminidase in human cortical nephron segments: heterogeneous changes in acute renal failure and acute rejection following kidney allotransplantation.

The catalytic activities of alkaline phosphatase and N-acetyl-beta-D-glucosaminidase, constituents of luminal brush-border membranes and lysosomes of kidney tubular cells, were measured in human kidney allografts in the maintenance and recovery phases of acute renal failure and in acute rejection crisis. The enzyme activities were fluorometrically determined in single microdissected cortical nephron segments of biopsies from 4 kidney allografts taken intraoperatively and postoperatively at different periods, which exhibited either good function or dysfunction. For comparison, the unaffected part of a human kidney nephrectomized due to hypernephroma as well as a biopsy of a morphologically normal human kidney were examined. Both enzymes displayed highest activities in the proximal part of the human nephron. In some intraoperative and postoperative biopsies with acute renal failure, alkaline phosphatase activity was reduced in proximal tubules, predominantly in the straight portion. This reduction could not be correlated with function. In acute rejection, very low alkaline phosphatase activities were uniformly found in proximal convoluted and straight tubules. Furthermore, intraoperative biopsies and biopsies of the functioning allograft have only approximately 50% of normal N-acetyl-beta-D-glucosaminidase activity in proximal convoluted tubules, but generally normal values in the straight portion. However, in acute renal failure, this enzyme activity was several-fold enhanced along the whole nephron, when compared with intraoperative values. In acute rejection, N-acetyl-beta-D-glucosaminidase activity was slightly reduced in proximal convoluted tubules, when compared with biopsies showing good function. It is suggested that the decrease of proximal tubular enzyme activities is the consequence of increased enzymuria and inadequate enzyme regeneration. On the other hand, the overshoot of N-acetyl-beta-D-glucosaminidase activity in the maintenance phase of acute renal failure appears to indicate increased degradative capacity, associated with cellular regeneration along the whole nephron.

Calf thymus alkaline phosphatase. II. Interaction with detergents

1. 1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. 2. The Stokes' radius ( r) of alkaline phosphatase in each detergent was measured by gel filtration. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N- alkylsulphates , the apparent size increased with alkyl chain length, with r = 6.4 nm (C 9) and r = 7.3 nm (C 12). Tween 20 failed to solubilise the enzyme. 3. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10–50% without substantially altering the K m for p- nitrophenylphosphate . The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33%; cholate, 76%). Alkylsulphates (C 8–2) had no significant inhibitory effect during 24 h at 23°C. 4. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholateinhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect.