Isoenzymes of blood serum alkaline phosphatase under experimental cholemia

The activity and isoenzymic spectrum of alkaline phosphatase of the blood serum and liver under cholemia caused by deoxycholic acid were compared in healthy animals and in animals with the affected liver. It is shown, that under conditions of the bile acids higher content in the organism due to deoxycholic acid, the total activity increases considerably and there appears an isoenzyme absent in the blood serum of healthy animals. Changes in the activity and isoenzymic spectrum of alkaline phosphatase under experimental cholemia developing against a background of the healthy and affected liver are characterized by certain peculiarities.     

Measurement of serum alkaline phosphatase

Alkaline phosphatase is a commonly requested enzyme test in clinical chemistry. However, the enzyme is not particularly substrate specific, which has led to a proliferation of methods for its analysis. It can exhibit a variable instability effect depending on the techniques required for its storage or analysis. Methods can also be highly dependent on sample isoenzyme distribution and reagent purity, leading to problems in the quality control of its analysis and in the comparison of results obtained from different methods. Alkaline phosphatase is not tissue specific and this may on occasion lead to uncertainty in the interpretation of its measured activity in blood serum. In recent years there has been a number of attempts to standardize methodology for this and other enzymes. Perhaps an alternative approach to the measurement of alkaline phosphatase activity will alleviate some of the problems encountered.     

Effect of magnesium on the properties of zinc alkaline phosphatase.

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.     

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.     

Zinc and magnesium content of alkaline phosphatase from Escherichia coli.

Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared.     

Isoenzymes of lactate dehydrogenase, alkaline phosphatase, creatine kinase. Possible use as tumor markers

The electrophoresis of serum isoenzymes in cancer pathology at different stages has enabled us to recognise for LDH4 and LDH5, characteristics of sensitivity in all stages, with positivity greater than 60% in the advanced and multiply metastasized stages or with interference from other pathologies; to reveal portions pathologically heavy like FAST (46%); to reveal atypical bands (BB and MIT), as well as the values for normal or really low enzymatic activity. Through the elevated sensitivity and the high percentage of positivity, the isoenzymes LDH4 and LDH5 can be considered tumour markers and superior in validity to the conventional markers.     

Sugar chain heterogeneity of bone and liver alkaline phosphatase in serum.

Fractionation of bone and liver alkaline phosphatase (EC 3.1.3.1; ALP) in serum by serial lectin affinity chromatography has demonstrated differences in the sugar chain structure of bone and liver ALP in serum from that previously reported in the corresponding tissues, with a lower content of high mannose or hybrid-type sugar chains and a higher content of biantennary complex-type chains. Furthermore, the bone and liver ALPs were found to differ in the latter with the bone fraction showing a greater content of fucose residues.     

Alkaline phosphatase in human lymphocytes. II. A method for ultracytochemical detection of alkaline phosphatase in lymphocytes

For the ultracytochemical identification of alkaline phosphatase in lymphocytes gained from the peripheral blood of healthy individuals a sensitive method is described which allows the low enzyme activity of these cells to be determined. This was possible because the authors succeeded in stabilizing lead ions in the alkaline medium by forming a complex directly between tris-(hydroxymethyl) aminomethan and lead (II) citrate. AP localized ultrachemically in lymphocytes in particular formations similar to phosphasomes of neutrophilic granulocytes. In those lymphocytes stimulated by lipopolysaccharides a high enzyme activity could be observed and, in addition to phosphasomes, the product of response can also be found in canal-like structures of the endoplasmatic reticulum. These findings contribute to clarify the ultrastructural localization of alkaline phosphatase in lymphocytes and may be regarded as an aid in discovering the importance of the enzyme in the biology of lymphocytes or in its activation, respectively.