Interaction between alkaline phosphatase and ascorbic acid by fluorescence and absorption studies

Very low amounts of ascorbic acid modify alkaline phosphatase fluorescence, absorption and enzymatic activity. A strong quenching of enzyme, tryptophan and tyrosine emission together with evident alterations of the protein absorption characteristics are observed. The catalytic activity inhibition probably reflects a perturbation of the active site environment due to the interaction of ascorbic acid with enzyme aminoacyl residues.     

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium

Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.     

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.     

Characterization of alkaline phosphatase in tissue sections by microspectrofluorometry

Synopsis Kinetic characteristics of alkaline phosphatase (EC 3.1.3.1) were determined in cryostat sections of rat kidney by microfluorometry with -naphthyl phophate as substrate, and the results were compared with measurements on enzyme extracted from this tissue. The apparent Michaelis constant of the enzyme in cryostat sections was found to be 0.6mM, in good agreement with the value of 0.8mM determined for the enzyme in solution. The pH-dependence of enzyme activity was also similar for the enzyme in the two states. These results suggest that release of alkaline phosphatase from its binding-sites during extraction and purification does not markedly alter its catalytic properties; also, the mutual agreement of histochemical and biochemical data give support to the validity of the histochemical technique.     

A kinetic approach to the evaluation of alkaline phosphatase inactivation by urea

A kinetic approach is described which enables the measurement of the enzyme inactivation rate constant during the reaction course. A mathematical analysis is presented and it is shown that a time-dependent step may be postulated to exist. Reaction kinetics follow an exponential rule with time as the independent variable and enzymatic activity as the dependent variable. A simple procedure of graphical analysis is reported and the influence on the inactivation rate constant of various conditions (temperature and inhibitor concentration) is evaluated. The method is illustrated by an experimental model: the inactivation of bovine kidney alkaline phosphatase by urea.     

Rapid induction of alkaline phosphatase activity by retinoic acid

Retinoic acid (vitamin A acid) increased alkaline phosphatase activity in cultured cells derived from both normal rat prostate and the Dunning R-3327 transplantable prostatic adenocarcinoma. Retinoic acid was found to be 3–4-fold more effective as an inducer of enzyme activity than retinol or retinal. In one rapidly-growing cell line (UMS-1541Q) which has a barely-detectable level of enzyme activity in the uninduced state, increased activity could be detected as early as 3–4 hours after the addition of 10μM retinoic acid. This increase was totally blocked by actinomycin D and cycloheximide. The demonstrated rapid inducibility of alkaline phosphatase activity provides a specific marker for the action of retinoic acid at the molecular level.     

Characterization of alkaline phosphatase in canine serum

On the basis of carbohydrate structure, normal dog serum contains three basic types of serum alkaline phosphatase (SAP) corresponding to (1) highly branched complex (non-concanavalin A-binding), (2) complex, or (3) high-mannose (both concanavalin A-binding) oligosaccharide structures. Subsequent binding experiments with monoclonal antibody to intestinal alkaline phosphatase (AP) and bromotetramisole inhibition studies clearly indicated the presence of intestinal-like SAP. Concanavalin A (Con-A) binding characteristics suggested the presence of a bone-like SAP. Con-A-binding and isoelectric focusing results revealed the presence of two (type Ib and IIb) major SAP isoenzymes thought to be of hepatic origin. SAP isoenzymes appear to be modified when compared to tissue AP, particularly in regard to molecular size and, in some cases, carbohydrate structure.     

Rapid inactivation of bacteriophage T7 by ascorbic acid is repairable

Treatment of bacteriophage T7 with ascorbic acid resulted in the rapid accumulation of single-strand breaks in the DNA with double-strand breaks appearing only after incubation times of 20 min or longer. The single-strand breaks were responsible for a rapid inactivation of the phage as assayed by immediate plating of the phage-bacteria mixture on nutrient agar. Incubation of the phage-bacteria mixture in liquid medium prior to plating allowed a host cell reactivation process to repair the nicks and reactivate the phage. Non-reversible inactivation of the phage was a slower process which could be correlated with the appearance of double-strand breaks in the phage DNA. Host cell reactivation of the phage was also manifested in the phenomena of delayed lysis and delayed appearance of the concatemeric DNA replication intermediate.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.     

Inhibition of alkaline phosphatase by thioureido derivatives of methylenebisphosphonic acid

A series of thioureido derivatives of methylenebisphosphonic acid were synthesized by the reaction of aminomethylenebisphosphonic acid with the corresponding isothiocyanates, and their effect on the activity of alkaline phosphatases from bovine small intestine mucosa (BSIM) and human placenta was studied. It was found that (3-phenylthioureido)methylenebisphosphonate is approximately one order of magnitude more effective in inhibiting the activity of alkaline phosphatase from BSIM than the alkyl derivatives of thioureidomethylenebisphosphonic acid with methyl, ethyl, tert-butyl, or cyclohexyl substituents. The introduction of substituents into the benzene ring of (3-phenylthioureido)methylenebisphosphonate decreased the effect of the inhibitor on the activity of the enzyme. The affinity of (3-phenylureido)methylenebisphosphonate to the alkaline phosphatase of BSIM was also weaker as compared with the corresponding thioureidomethylenebisphosphonate. The insertion of thioureidobisphosphonates into the active site of alkaline phosphatase of human placenta by the method of molecular docking indicated that the methylenebisphosphonate residue and the substituted amino groups of the inhibitor are involved in the mechanisms of complex formation with the enzyme. It is supposed that the improvement of the inhibitory activity of (3-phenylthioureido)methylenebisphosphonate toward alkaline phosphatase of BSIM is due to the additional fixation of the phenyl substituent in the active site of the enzyme.     

Inhibition of alkaline phosphatase by thioureido derivatives of methylenebisphosphonic acid

A series of thioureido derivatives of methylenebisphosphonic acid were synthesized by the reaction of aminomethylenebisphosphonic acid with the corresponding isothiocyanates, and their effect on the activity of alkaline phosphatases from bovine small intestine mucosa (BSIM) and human placenta was studied. It was found that (3-phenylthioureido)methylenebisphosphonate is approximately one order of magnitude more effective in inhibiting the activity of alkaline phosphatase from BSIM than the alkyl derivatives of thioureidomethylenebisphosphonic acid with methyl, ethyl, tert-butyl, or cyclohexyl substituents. The introduction of substituents into the benzene ring of (3-phenylthioureido)methylenebisphosphonate decreased the effect of the inhibitor on the activity of the enzyme. The affinity of (3-phenylureido)methylenebisphosphonate to the alkaline phosphatase of BSIM was also weaker as compared with the corresponding thioureidomethylenebisphosphonate. The insertion of thioureidobisphosphonates into the active site of alkaline phosphatase of human placenta by the method of molecular docking indicated that the methylenebisphosphonate residue and the substituted amino groups of the inhibitor are involved in the mechanisms of complex formation with the enzyme. It is supposed that the improvement of the inhibitory activity of (3-phenylthioureido)methylenebisphosphonate toward alkaline phosphatase of BSIM is due to the additional fixation of the phenyl substituent in the active site of the enzyme.     

Stabilization of human serum alkaline phosphatase to histidine-induced heat inactivation by tryptic digestion.

1. Serum alkaline phosphatase [EC 3.1.3.1] was strongly inactivated by histidine during incubation at pH 8.0 and 45degrees; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further. 2. The effect of trypsin on the histidine-induced heat inactivation differed considerably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.     

Characterization of alkaline phosphatase from bovine polymorphonuclear neutrophils

We characterized the bovine polymorphonuclear neutrophil alkaline phosphatase which was considerably purified with a sp. act. of 206 units/mg of protein. The Km value for p-nitrophenylphosphate at pH 10.0 was 1.69 mM. L-Histidine, imidazole and L-homoarginine but not L-phenylalanine inhibited the enzyme. In heat stability study, the enzyme lost 50% activity at 56 degrees C for 20 min. The enzyme had a half-life of 30 min in 3 M urea at 37 degrees C and pH 7.5. The enzyme was inhibited by beta-mercaptoethanol in a dose-dependent fashion. It is suggested from above results that the neutrophil alkaline phosphatase isozyme could be distinguishable from other tissue isozymes.     

Ethylenediamine-NN''-tetra-acetate-dependent amino acid-stimulated inactivation of mouse ovarian alkaline phosphatase activity.

1. Preincubation of partially purified preparations of mouse ovarian alkaline phosphatase in the presence of both EDTA and glycine at alkaline pH resulted in a pronounced inactivation of alkaline phosphatase activity. Inactivation did not occur on preincubation with EDTA or glycine alone. 2. The rate of inactivation was first-order with respect to the concentration of enzyme, and was independent of EDTA concentration above a threshold value. 3. The process was pH-dependent with a pK at 9.85, and inactivation was not dependent on the stereochemistry of the amino acid. A free alpha-amino group and a free carboxyl group at a specific spatial separation were essential for inactivation. 4. Inactivation involved the formation of an enzyme--metal ion--amino acid complex, the amount formed being dependent on both the nature and concentration of the amino acid. This complex then decayed to a derivative that was then acted on by EDTA, yielding an inactive form of the enzyme.     

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.     

Characterization ofZymomonas mobilis alkaline phosphatase activity inEscherichia coli

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed inEscherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42°C. This enzyme required Zn²⁺ for its catalytic activity; however, Mg²⁺ or Ca²⁺ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn²⁺. Kinetics ofZ. mobilis alkaline phosphatase production inE. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.     

Characterization of chicken plasma alkaline phosphatase isozymes by urea and heat treatments

The effects of urea and heat treatments on electrophoretic pattern and activity were investigated in chicken plasma alkaline phosphatase (AP). The B band, which had a slower migrating rate to the F or S band irrespective of isozyme types, was labile to urea (4M) and heat treatments (60 ^oC, 10 min), while the F and S bands were stable to the same treatments. From these results, the genetic control of the three chicken plasma AP isozymes, i.e., F, S and B bands, was discussed.
The total AP activity of the F or S type was little affected by urea treatment in spite of the unstableness of the B band. It is considered that the B band inactivated by urea restores the activity when the urea concentration was reduced. The AP activity was reduced by the heat treatment. The reduction may be primarily due to the loss of the activity of the B band.