Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines (original) (raw)
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Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.
Expression of intestinal alkaline phosphatase in human organs
Histochemistry, 1992
Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.
Intestinal-like alkaline phosphatase expressed in normal human adult kidney
European journal of biochemistry / FEBS, 1989
Human adult kidney was found to contain not only the 'tissue-unspecific alkaline phosphatase' but also another alkaline phosphatase isozyme. By use of monoclonal antibodies specific for human intestinal alkaline phosphatase, this kidney isozyme was purified to homogeneity by immunoaffinity chromatography. The structural and kinetic properties of the enzyme were compared with those of the other alkaline phosphatase isozymes expressed in normal human tissues, i.e. the placental, intestinal, meconial (fetal), liver and kidney isozymes. The new kidney isozyme was clearly different from both the tissue-unspecific and the adult intestinal alkaline phosphatase as regards isoelectric point, molecular mass and peptide maps after cyanogen bromide cleavage, but it was found to be identical to the meconial alkaline phosphatase. The results demonstrate simultaneous expression of two alkaline phosphatase isozymes in human kidney, one of which is normally related only to the fetal intestine.
Expression of heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells
岐阜藥科大學紀要, 1995
A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.
Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes
Biochemical Journal, 1979
The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all ot...
Molecular properties of rat intestinal alkaline phosphatase
Biochim Biophys Acta Protein, 1976
Rat intestinal alkaline phosphatase is an heterogeneous glycoprotein that contains three protein sub-forms separable by electrophoresis. The molecular weight for the glycoprotein (i.e. the average for the three sub-forms) is 157 000-160 000. Three protein sub-forms are detectable on sodium dodecyl sulphate-polyacrylamide gel electrophoresis that migrate at rates corresponding with molecular weights of 64 000, 79 000 and 92 000. Treatment of native alkaline phosphatase with 6 M guanidine. HCI or buffer at pH 3.0 results in a product with a molecular weight of 78 000 and 70 000, respectively. Thus it is concluded that each of the three sub-forms is a dimer of identical or closely similar subunits. Limited proteolysis results in the production of new enzymically active sub-forms separable by electrophoresis. Using a bacterial protease it is possible to convect intestinal alkaline phosphatase into a form with a molecular weight of 132 000 without causing any significant change in kinetic properties. Electrophoresis of this new form on sodium dodecyl sulphate polyacrylamide gel suggests that it is composed of 66 000-dalton subunits. The native enzyme contains at least 20 ~ by weight of carbohydrate that probably contributes to microheterogeneity of a second degree superimposed on that stemming from the presence of three protein sub-forms. Treatment with various glycosidases has no effect on electrophoretic behaviour, however. It is suggested that the three sub-forms possibly represent different stages of a maturation process that operates by limited proteolysis of a single parent protein.
Journal of Biological Chemistry
In the suckling rat intestine, alkaline phosphatase is present in both soluble and membranous forms. These forms were purified from the 105,000 X g ileal supernatant and jejunal particulate fractions, respectively. Protease inhibitors added to the homogenate buffer did not significantly alter the yield of soluble and membranous enzyme. Moreover, cytoplasmic fractions from suckling rat ileum containing active lysosomal enzymes degraded rather than solubilized the membranous form. The two forms were found to be kinetically similar and immunologically identical. The soluble form had M, = 108,000 in nondenaturing sodium dodecyl sulfate gels and a single subunit in denaturing sodium dodecyl sulfate gels with M, = 47,000. Comparable values for the membranous forms were M, = 130,000 and 66,000. With Triton polyacrylamide gel electrophoresis, the RF of the soluble form was 0.48, faster than the membranous form (RF 0.20). These values were identical for crude and purified enzymes. Analysis of carbohydrate content revealed that the soluble form contained more fucose (20 versus 8 mol of sugar/g of protein X lo-') and N-acetylgalactosamine (32 versus 14 mol of sugar/g of protein X lo-') than did the membranous form. The membranous enzyme displayed strong lipid affinity by ready incorporation into liposomes, while the soluble form failed to be included. The two enzyme forms had different patterns of molecular heterogeneity by isoelectric focusing, with bands of activity at pH 4.97 and 5.06 for the soluble form and 4.33, 4.43, and 4.60 for the membranous enzyme. The papainsolubilized membranous enzyme migrated similarly to the soluble form on Triton-and sodium dodecyl sulfate gel-electrophoresis but reproduced the pattern of the membranous form on isoelectric focusing. From these studies, we conclude that: (a) the soluble and membranous forms of intestinal alkaline phosphatase are closely related but distinct isozymes; and (b) the soluble form is not simply a proteolytically solubilized product of the membranous enzyme.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1981
In the small bowel mucosa of the rot, alkaline phosphatase was found to be present in the 105 000 × g supernatant fraction, in addition to the major brush border membrane-bound form. This soluble enzyme contributed up to 3.6% of total alkaline phosphatase activity in the adult rat. Combined use of inhibitors of serine, thiol and carboxyl proteases during homogenization did not significantly affect the proportion of enzyme activity in the supernatant fraction. In suckling rats a much larger proportion of alkaline phosphatase was soluble, reaching 36.5% in the 14-day-old animal. The soluble and membranous forms were compared in adult and suckling rats by examining their biochemical and immunological characteristics. In the adult rat the specific activity of membranebound alkaline phosphatase showed a progressive fall distal to the duodenum, while maximal specific activity of the supernatant enzyme occurred in the distal ileum. By contrast, in the suckling rat the specific activity of both forms increased from the duodenum to the ileum. Soluble alkaline phosphatases in mature and immature rats were distinct from the membranous forms when examined by polyacrylamide gel electrophoresis in the presence of Triton X-100. Electrophoretic heterogeneity was seen in the soluble adult enzyme, characteristic Rf values occurring for each level of the small bowel (i.e., duodenum, jejunum and ileum). Such heterogeneity was not seen in suckling rats. Antigenically, the soluble and membranous forms were identical to each other in adult and suckling rats. Analysis of mucosal cells isolated sequentially from tip of the villus to crypt showed that both the soluble and membranous isozymes had highest activity towards the tip of the villus. We conclude that the soluble and membranous forms are distinct biochemically and yet are antigenically identical and arise from the same level of the villus. The presence of the soluble form, which is not an artifact of tissue preparation, indicates that alkaline phosphatase is not exclusively an integral membrane protein.
Tumor Biology, 1991
Using alkaline phosphatase isozyme-specific immunocatalytical assays, the content of isozymes was determined in normal mucosas and adenocarcinomas from human colon or rectum. Tumor levels of both the tissue (liver)-unspecific and the placental-like alkaline phosphatase (PLAP-like) were elevated compared to normal mucosas of the same patients. Such elevations have been reported previously, particularly in seminomas and ovarian tumors. In several tumors, moreover, the intestinal isozyme was expressed in lesser amounts than in the adjacent mucosa. The present results indicate that the activation of two of the phosphatase isozymes, including expression of the typical germ cell line phosphatase (the PLAP-like isozyme), may occur even in nongonadal tumors. This may reflect an induction pattern of phosphatase isozymes, with implications for malignant transformation also in other tumors.