Norma R. Inglis

Distinctions between intestinal and placental isoenzymes of alkaline phosphatase

Abstract 1. 1. Placental and intestinal alkaline phosphatases are equally inhibited by 0.005 M l -phenylalanine, a fact which complicates the interpretation of the values for l -phenylalanine-sensitive alkaline phosphatase of pregnancy serum which can be expected to have both phosphatases. 2. 2. Contrary to (human and rat) intestinal alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) the isoenzymes of human and rat placental tissues are split by neuraminidase ( N -acetyl neuraminate glycohydrolase, EC 3.2.1.18) i.e. the anodic mobilities of the placental enzymes are appreciably reduced by prior incubation with neuraminidase. 3. 3. Advantage is also taken of the marked heat-stability of placental alkaline phosphatase and thus starch gel electrophoretic patterns of the placental and intestinal enzymes in heated and unheated specimens are different from each other. 4. 4. The pH optima of human intestinal and placental alkaline phosphatases are 9.8 and 10.6 with 18 m M phenylphosphate as substrate. The ratio of the enzyme activity at pH 10.6 to that at pH 9.8 is ten times higher for placenta than that for intestine (1.1 versus 0.11) with 2 m M phenylphosphate as substrate. 5. 5. Although the Michaelis constants of placental and intestinal alkaline phosphatase at pH 10.6 are the same, the K m -values of placental alkaline phosphatase at lower pHs are considerably higher than those of the intestinal isoenzymes. 6. 6. The above electrophoretic and kinetic differences may be employed to distinguish from each other human intestinal and placental alkaline phosphatase isoenzymes and may be utilized further to investigate the intestinal and placental origins of serum alkaline phosphatase.

Serum alkaline phosphatase in hypophosphatasia

It is recognized that serum alkaline phosphatase may reflect enzyme contributions from bone, liver, and intestine. We have investigated serum alkaline phosphatases in two siblings with hypophosphatasia. After administration of long-chain triglycerides, the major alkaline phosphatase component of their sera was shown to be of intestinal origin on the basis of inhibition by l-phenylalanine. Starch block electrophoresis suggested that there were other regions of l-phenylalanine-sensitive alkaline phosphatase in addition to the major slow-moving intestinal band. Medium-chain triglycerides which are absorbed by the portal route did not cause a similar augmentation of intestinal alkaline phosphatase activity. These studies indicate that serum levels of intestinal alkaline phosphatase are increased normally after long-chain fat feeding in hypophosphatasia and may be the major component of total serum alkaline phosphatase activity.

Preparation and characterization of human intestinal alkaline phosphatase antigens

Abstract A simple procedure is outlined for the preparation of human intestinal alkaline phosphatase of sufficient purity for use as an antigen. Two molecular weight species (variants A and B) were isolated with high and low saturations of ammonium sulfate. The lower molecular weight fraction. Variant A, was further purified by Sephadex G-200 filtration. The various fractions were characterized by studying enzyme activity in the presence and absence of magnesium ions, l -phenylalanine, l -homoarginine, and by measuring their sensitivity to heat. In addition, electrophoretic patterns were studied on starch gel. Purified enzyme was injected intradermally into rabbits and the resultant antiserum was used to identify alkaline phosphatase isozyme bands of human chyle, serum and bowel juice when migrated on starch gel.

Preparation of two antigens of human liver isoenzymes of alkaline phosphatase

Abstract A convenient standard technique has been developed to prepare human liver isoenzyme (alkaline phosphatase) antigens based on an initial butanol treatment of a tris-homogenate of liver, followed by acetone precipitation, fractionation with ammoniumzsulfate and Sephadex G-200 gel filitration. The bulk of the enzyme was distributed in a bimodal manner, one moiety separating out in 0–30% saturated ammonium sulfate solution and the other, in 50–60% saturated ammonium sulfate. The latter, after Sephadex gel filtration is designated variant A and the former, variant B. Both variants have been characterized on starch gel electrophoresis and with respect to sensitivity to Mg2+, l -phenylalanine and l -homoarginine. Variant A (mol. wt. 280000) corresponds to the fast liver band (6 cm) seen in sera rich in liver isoenzyme and variant B mainly with the isozyme band at or near the origin. These two variants represent two characterized liver antigens capable of producing the desired specific antisera.

Convenient immunofixation electrophoresis on cellulose acetate membrane

Abstract Applying polyspecific antiserum onto cellulose acetate membrane after electrophoretic separation can result in a simultaneous fixation of multiple antigens as discrete bands with a resolving power of at least 30 ng protein per band.

A comparison of chyle isoenzymes of alkaline phosphatase in chyle and hypophosphatasemic sera

Abstract A chylous effusion fluid was the source of “chyle” alkaline phosphatase which has been partially purified. It has been characterized with respect to migration on starch gel, effect of neuraminidase, reaction with rabbit antisera to liver and to intestinal isoenzymes and with regard to its inhibition by l -phenylalanine and l -homoarginine. The major component has been identified as intestinal-type alkaline phosphatase and a minor component has the properties of liver-type alkaline phosphatase. The identical results were obtained in the examination of the sera of two hypophosphatasemic children which suggests a major chylous source of the alkaline phosphatase in hypophosphatasemia.

Sephadex G-200 gel electrophoresis of human serum alkaline phosphatases

Abstract A method for the separation of the variants of human alkaline phosphatase by horizontal Sephadex gel electrophoresis has been developed. The electrophoresis was earried out with gels containing 3% Sephadex G-200 of particle size 10–40 μ. The localization of enzyme and protein bands was successful on Sephadex gel, especially with highly purifled enzyme samples of very low protein concentrations (50 μg). A much more rapid 4 hr method has been devised also for thin-layer Sephadex G-200 gel electrophoresis which produces a dry transparent film of great tensile strength suitable for a permanent record. These new methods have been successfully applied to separate the components of alkaline phosphatase in sera of normal and some hyperphosphatasemic subjects.