Placental Isoenzyme Research Articles

Differential release of membrane-bound alkaline phosphatase isoenzymes from tumor cells by bromelain

A rapid enzymatic method is presented which results in the selective release of cell-surface alkaline phosphatase isoenzymes. Treatment of suspensions of human tumor cell lines with the proteolytic enzyme bromelain released certain alkaline phosphatase isoenzymes into low-molecular-weight, catalytically active forms. Cells which expressed term placental or intestinal isoenzymes were equally susceptible to this treatment. A cell line which expressed early placental isoenzyme, however, was unaffected by bromelain as indicated by complete recovery of activity in the treated membrane fraction. Successful solubilization of immunologically reactive enzyme allows quantitation of levels of cell-surface enzyme in response to modulators of gene expression. Moreover, the observed selective solubilization of isoenzymes by bromelain may be of general use in analyses of the physical association between other biologically important surface proteins and the lipid components of the cell membrane.

Occurrence of heat-labile Regan type of alkaline phosphatase in hematopoietic tumors.

The physiocochemical and immunological properties of alkaline phosphatase extracted from Hodgkin's nodes, non-Hodgkin's lymphoma nodes and leukemic leukocytes have been studied. The alkaline phosphatase from these three tumor types possesses the same biophysical and biochemical properties and immunological determinants as the placental alkaline phosphatase. However, it is more heat-labile than the placental isoenzyme. Immunological experiments indicate that, of these tumor types, Hodgkin's tumor contains the largest amounts of heat-labile Regan type of alkaline phosphatase.

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.

Alkaline phosphatase isoenzyme expression in chang liver cells

Chang liver cells (ATCC, CCL 13) are shown to express three distinct isoenzymes of alkaline phosphatase. One of these is identical to liver alkaline phosphatase by all criteria. The other two isoenzymes antigenically resemble placental alkaline phosphatase, and one is physically identical to this latter isoenzyme. The third phosphatase differs in isoelectric point and subunit molecular weight from the term placental isoenzyme but closely resembles a variant isoenzyme previously described in human KB cells [8]. We have examined the expression of these three catalytically similar enzymes in a series of single cell clones derived from this cell line. Our results demonstrate that (1) all three isoenzymes can be coincidentally expressed by the progeny of a single cell; and (2) the expression of the liver and placental isoenzymes vary independently of one another while that of the variant placental enzyme is dependent upon the expression of the normal placental type.

Demonstration of placental alkaline phosphatase in human breast cancer.

The screening of a series of 11 metastatic breast tumors for the presence of the placental isoenzyme of alkaline phosphatase (EC3.1.3.1) by RIA revealed one strong producer. The alkaline phosphatase of this tumor was characterized with respect to its immunochemical cross-reactivity, inhibition by L-phenylalanine and levamisole, subunit molecular weight (Mr) and isoelectric point (pl) in two-dimensional electrophoresis, and one-dimensional peptide map. In all parameters of the characterization, the tumor alkaline phosphatase, except for subunit molecular weight which was slightly lower (60,000 versus 64,000 for the placental isoenzyme). No strong placental alkaline phosphatase producers were found among 16 primary tumors examined by RIA. The screening of patients' sera for the placental alkaline phosphatase using RIA indicated elevated levels over post-menopausal controls in 20% of the metastatic patients. Only 3% of the primary patients had elevated serum levels. These results suggest that the placental isoenzyme of alkaline phosphatase may be a useful tumor marker for recurrent breast cancer.

Sulfonamide inhibition of human alkaline phosphatase

Several sulfonamides used as antibacterial or diuretic drugs are potent alkaline phosphatase inhibitors. The mechanism of inhibition may involve binding of the zinc in the active site of the enzyme by the unbonded electron pair on the sulfonamide group nitrogen atom as well as binding of the drug to a second site. Addition of progressively larger groups to this nitrogen leads to an increasing loss of inhibitory capability. Isoenzymes from human liver, bone, kidney, granulation tissue and intestine are inhibited to a similar extent while the placental isoenzyme is more resistant, It is suggested that some pharmacologic actions of sulfonamides may be due to inhibition of alkaline phosphatase, rather than carbonic anhydrase.

The effect of sucrose and other carbohydrates on human alkaline phosphatase isoenzyme activity

The modification of alkaline phosphatase (ALP) activity by ten carbohydrates ( d-isomers) was studied. These included three disaccharides : sucrose, lactose and maltose; four hexoses: glucose, galactose, mannose and fructose; and three pentoses: arabinose, ribose and xylose. In all cases the inhibition of placental isoenzyme activity was in proportion to the concentration of carbohydrate. Intestinal and hepatic ALP was activated at low carbohydrate concentrations and inhibited at high concentration. The exception was maltose which inhibited hepatic alkaline phosphatase markedly at all concentrations. Lineweaver-Burk plots generated by kinetic studies at pH 9.8, 10.15, 10.5 and 10.7 showed a mixed-type inhibition. With the use of sucrose in the assay medium it was possible to distinguish two types of term placental-like ALP isoenzymes of tumor origin, which differ in their sensitivity to inhibition by l-leucine. The Nagao type ( d-leucine sensitive) was inhibited more by sucrose than was the non- d-leucine sensitive isoenzyme. The inhibitory effect of carbohydrates on placental ALP activity was independent of pH and substrate concentration, unlike the modifying effect on the activity of intestinal and hepatic enzyme by the carbohydrates studied. It is suggested that carbohydrate-rich media may accentuate conformational differences of the various isoenzymes with consequent effects on the availability of the active sites to the substrate.

Aktivitätsbestimmung des placentalen Isoenzyms der alkalischen Phosphatase im Serum: Ein Methodenvergleich

Three methods were compared in human serum for determining the activity of the placental isoenzyme of alkaline phosphatase: 1. Heat inactivation at 65 degrees C for 10 minutes, 2. Differential inactivation with L-p-Bromotetramisole, and 3. an immunological precipation test. A good comparison between the three methods was found (correlation coefficient between 0.973 and 0.982). For long series or screening determinations for "Regan-isoenzyme" the differential inactivation with L-p-Bromotetramisole is preferred because of the short analysis time and the possibility of simple mechanisation.

Human placental cell surface antigens: Expression by cultured cells of diverse phenotypic origin

The present work examined the expression of cell surface glycoprotein antigens in cultured human cell lines. The set of glycoproteins studied was defined by their immunoreactivity with antiserum developed to Triton-solubilized extracts of placental brush border membranes. Studies were performed using cell lines of trophoblastic (BeWo, JEG-3) and nontrophoblastic (Chang liver cells) origin, as well as diploid fibroblast cell lines (WI-38, GM-38). Antiplacental brush border antiserum reacts with at least 19 distinct antigens present in placental membrane preparations, each of which can be resolved and identified in two-dimensional electrophoresis. The subunit molecular weight and isoelectric point for all components were defined by their positions in the two-dimensional matrix. Thirteen of these could be detected among the five cell lines examined by lactoperoxidase-catalyzed cell surface iodination. One of these 13 antigens has been identified as the placental isoenzyme of alkaline phosphatase (PAP). The expression of this component is limited to choriocarcinoma cells and Chang liver cells and it is not present in diploid fibroblasts. Under normal circumstances expression of PAP is unique to the differentiated placenta but has been frequently demonstrated in both trophoblastic and nontrophoblastic neoplasms. Two other antigens are variably expressed among the different cell types examined in the present study and their presence or absence was independent of the trophoblastic, epithelial nontrophoblastic, or fibroblastic origin of the cells. Ten surface antigens were expressed in all five cell lines. Six of these had previously been found common to membranes from three adult differentiated tissues, including liver and kidney, as well as placenta (Wada et al, J Supramol Struc 10(3): 287-305, 1979). The presence of this set of antigens in cultured cells as well extends the possibility that these are ubiquitously expressed on human cell surfaces. Two other antigens observed in all cultured cells had been found in both placental and either kidney or liver membranes and may represent common functions shared by many tissues which are also necessary for growth in vitro. The two remaining placental antigens seen in all cultured cells have previously been shown to be absent in adult tissues. Their presence in cultured cells but not in the membranes of resting differentiated tissues may signify the expression of glycoproteins characteristic of trophoblasts in all cells adapted to growth in culture.

Modulation of HeLa Cell Alkaline Phosphatase Isoenzyme Expression by Prednisolone

Various HeLa cell lines have been examined in order to determine the effect of hormone treatment upon alkaline phosphatase isoenzyme expression. Both biochemical and immunoelectrophoretic analyses show that three cell lines demonstrating activity of the placental‐type (Regan) isoenzyme in absence of prednisolone are induced to much higher levels after growth in hormone‐containing medium for 72 hours. Prednisolone‐treatment resulted in increased enzyme activity but no change in the isoenzyme profile as determined by microzone and polyacrylamide disc gel electrophoresis. On the other hand, the majority of the activity in HeLa line D98AH2 in absence of hormone was found to be heat‐sensitive and inhibited by phenylalanine. Immunoelectrophoretic analysis indicated that amnion/intestinal isoenzyme was expressed. No appreciable change in total enzyme activity occurred in presence of hormone despite the induction of the Regan isoenzyme and the diminution of amnion/intestinal type. Thus, treatment of this cell line with hormone resulted in differential expression of the amnion/intestinal and placental isoenzyme components. These results support a regulatory function of prednisolone with respect to expression of alkaline phosphatase isoenzymes in tumor cell lines and identify a HeLa cell line, D98AH2, which elaborates the amnion/intestinal isoenzyme.

Occurrence in Normal Human Testis of a Heat‐stable Alkaline Phosphatase Similar to a Rare Variant of Placental Isoenzyme

The possibility that testicular germ cells are ‘totipotential’ in expressing early developmental gene products was examined in the case of placenta‐specific heat‐stable alkaline phosphatase. In each of the 11 adult testes studied about 0.3–4.6% of the total alkaline phosphatase activity was heat‐stable and L‐phenylalanine‐sensitive but L‐homoarginine‐insensitive. However, the testicular heat‐stable enzyme was more susceptible to inhibition by L‐leucine and EDTA than the normal placental and intestinal enzymes, thus resembling the so‐called ‘D‐variant’ of placental alkaline phosphatase. By antibody‐directed enzyme inhibition tests the testicular heat‐stable enzyme crossreacted completely with normal placental enzyme but clearly distinguished itself from a heat‐stable component of normal intestinal enzyme. Thus, a minute amount of placental alkaline phosphatase D‐variant is synthesized in testis, indicating that the gene for elaborating this placental protein is probably already active in the germ cells of testis. The high incidence of this protein in cancers of testis and ovary is probably due to the increased production of this protein by active gonadal genes.

Identification of placental alkaline phosphatase in human sera using polyacrylamide gel electrophoresis

Polyacrylamide gradient gel electrophoresis may be used to separate placental alkaline phosphatase from other types of serum alkaline phosphatase. This method, of which specificity is demonstrated by the thermostability and immunological reaction of the placental form, is sensitive enough to detect an activity of 0.02 mU in a sample, and seems suitable for use in investigating placental isoenzyme in serum.

Regan variant alkaline phosphatase in gastrointestinal carcinoma

A fast-moving alkaline phosphatase band on polyacrylamide gel electrophoresis has been found in 6 patients with carcinoma of the liver and gastrointestinal tract. This isoenzyme resembled the placental isoenzyme in its inhibition by L-phenylalanine, its resistance to L-homoarginine inhibition and its molecular weight. However, it differed from the placental and Regan isoenzymes in its sensitivity to L-leucine and ethylenediaminetetra-acetic acid, its lower retardation by neuraminidase, its electrophoretic mobility and its decreased heat stability. The latter two properties also distinguished it from the Nagao isoenzyme. It was identified as the Regan Variant. The Regan Variant has hitherto been reported largely in hepatocellular carcinoma. In the present paper we report its appearance in the sera of patients who have neoplasms in a variety of primary sites in the gastrointestinal tract. It is emphasized that, while the presence of the Regan Variant in serum may be taken as evidence of carcinoma, no conclusions can be drawn as to the site of the disease.

Characteristics of the inhibition of serum alkaline phosphatase by theophylline

1. The effect of various parameters on the inhibition of alkaline phosphatase activity by theophylline was studied. The influence of pH is great, being optimum for a value of 9.4. The temperature and the magnesium ion concentration have no to low effect. 2. The nature of the substrate and the nature of the buffer in which the enzymatic activity is measured have an effect:by measuring the activity of alkaline phosphatase in serum by a method with phenolphthaleinphosphate in Tris buffer, a negligible interference by therapeutic or toxic levels of theophylline is observed. 3. The inhibition by theophylline varies greatly with the origin of alkaline phosphatase: the liver isoenzyme is strongly inhibited, the intestinal one to a lesser degree and the placental isoenzyme almost not inhibited. 4. The inhibition of the liver and intestinal isoenzymes are uncompetitive and the inhibition constants were measured.

Effects of sodium butyrate on synthesis of human chorionic gonadotrophin in trophoblastic and non-trophoblastic tumours

SOME human tumours and tumour-derived cell lines are capable of producing ‘eutopic’ peptide hormones and other proteins that are normally synthesised by the tissue from which the tumour originated. For example, choriocarcinoma cells, which are derived from cancer of the placental trophoblast, can synthesise human chorionic gonadotrophin (hCG) (refs 1, 2), and cultured pituitary adenomas can produce growth hormone (GH) (ref. 3). On the other hand, it is well established that a variety of malignant human tumours can produce ‘ectopic’ peptide hormones and other proteins which are not normally associated with the tissue of origin4–8. Ectopic synthesis of placental proteins, including the placental isoenzyme of alkaline phosphatase as well as the hormones hCG and human placental lactogen, may provide specific markers for neoplasm, since these proteins are not normally found in measurable amounts in the serum of men or of non-pregnant women9. Several human glycoprotein trophic hormones, including hCG, thyroid-stimulating hormone (TSH), luteinising hormone (LH), and follicle-stimulating hormone (FSH), are composed of two non-identical subunits, α and β (refs 10–13). These four hormones have virtually identical α subunits; in contrast each has a unique β subunit, which is responsible for biological specificity12. Complete hCG and free subunits are secreted by normal and neoplastic placenta1,2,14 and by many non-trophoblastic tumours15,16. Since hCG is a eutopic hormone for trophoblastic tumours, but an ectopic hormone for non-trophoblastic tumours, it occured to us that its synthesis by the two types of tumours might be regulated differently. We therefore compared the synthesis of hCG and hCG-α by three choriocarcinoma cell lines—BeWo (ref. 1), JEG-3 (ref. 2) and Reid (P. O. Kohler, unpublished observations)—with the synthesis by two non-trophoblastic tumour-derived cell lines—ChaGo and HeLa. (ChaGo was derived from an hCG-producing pulmonary carcinoma16, and HeLa from a carcinoma of the cervix17.) We also tested the effects of sodium butyrate on the synthesis, since this compound had been shown to induce hCG-α and hCG synthesis in certain HeLa cells18. We found that in the absence of inducer, all of the cell lines can produce hCG-α—the trophoblastic tumour cells at roughly comparable rate, but the non-trophoblastic tumour cells at widely different rates (Fig. 1).

The quality control of alkaline phosphatase determinations with placental phosphatase

Human placenta has been recommended as the source of the alkaline phosphatase used for the preparation of control or reference materials. The effect of certain inhibitors, which may be present in the reagents, on control materials containing the placental isoenzyme, is shown to differ significantly from their effect on the isoenzymes in patient's sera. This finding indicates that if control materials are used which contain only human placental alkaline phosphatase, changes in accuracy resulting from differences in reagent quality may be missed, or alternatively a change in accuracy may be indicated by the quality control results which does not apply to the patient's sera.

L-p-Bromotetramisole, a new reagent for use in measuring placental or intestinal isoenzymes of alkaline phosphatase in human serum.

L-p-Bromotetramisole is proposed as a new reagent for use in determining intestinal and (or) placental isoenzymes of alkaline phosphatase in human serum. Its main advantage over L-phenylalanine is its high discriminating potency at very low concentrations. For highest accuracy, the samples may even be "titrated" with the inhibitor. Analytical recoveries of intestinal and placental isoenzymes in serum are complete, and results correlate well with those by other methods. At the appropriate concentrations, L-p-bromotetramisole gives nearly identical results on a variety of sera, irrespective of the buffer used. Within-day CV's were 6.1 and 2.1 for 20% and 60% of the resistant isoenzyme activity, respectively. The day-to-day CV was 3.45 for a sample containing 53.3% of the resistant isoenzyme (n=15). The well-known increase in placental isoenzyme activity with time of gestation could be confirmed on 612 sera from pregnant women. The activities of the intestinal isoenzyme in sera from healty blood donors (n = 126) and hospitalized patients (n = 135) agree with previous findings obtained by the other techniques.

Characteristics of alkaline phosphatase from two continuous lines of human choriocarcinoma cells

Several characteristics of human choriocarcinoma alkaline phosphatase have been studied. BeWo and Jar choriocarcinoma lines each contain heat-stable alkaline phosphatase activity with characteristics similar to the D-variant term placental isoenzyme. The activity is also similar to the Nagao enzyme found in many malignancies. Each choriocarcinoma also has heat-labile alkaline phosphatase activity with characteristics similar to the enzyme found in normal first-trimester placenta. Neither BeWo alkaline phosphatase could be stimulated with cortisol. Both BeWo alkaline phosphatase activities could be stimulated with methotrexate and actinomycin D.

Alkaline phosphatase. II. Conditions affecting determination of total activity in serum.

Results of previous experiments on isolated purified isoenzymes of alkaline phosphatase from humans have been confirmed on sera containing relatively large activities of the different isoenzymes. The most remarkable finding is that activation by N-ethylaminoethanol is much more pronounced, in the case of the intestinal and placental isoenzymes, than is activation by diethanolamine. For several reasons, it is suggested that N-ethylaminoethanol is the buffer of choice, 0.1 mol/liter concentration for routine measurements and 1 mol/liter in those cases where the determination of the intestinal or placental isoenzymes is important. Mg2+ could be omitted because its addition increases the activity only marginally. Normal values for adults with use of 0.1 mol/liter N-ethylaminoethanol are 59 +/- 36 (2 SD) U/liter (n = 126).

Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability.

Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.