Separation of alkaline phosphatase enzymes in human serum using gel-filtration (Sephadex G-200) techniques

Abstract

With the aid of gel filtration techniques (Sephadex G‐200) the alkaline phosphatase (Bessey‐Lowry‐Brock) distribution patterns have been studied in the serum of control human subjects and of those with conditions known to affect total enzyme levels. Protein was scanned at 254 and 280 mμ and the 19S, 7S, and 4S peaks thus obtained were used as points of reference. The characteristic control serum revealed one sharp peak of enzyme activity in the 7S protein region. A similar but sharper peak was found in osteomalacia and Pagets' disease. In contrast, in malignant bone conditions 10.4%, in metastatic malignant liver disease 31.36% and in nonmalignant liver disease 12.7% of total enzyme moved with the 19S proteins. The patterns found in various conditions are reproducible, and that of metastatic malignant liver disease is particularly characteristic. The significance of the macromolecular‐moving enzyme is discussed and is being investigated.

gamma-Glutamyl transferase isoenzymes in human bile.

“…There are several possible explanations for the existence of isoenzymes, and in this context it is now well established that enzymes may be attached to lipid or lipoprotein components (Moss, 1962;Dunne et al, 1967;Jennings et al, 1970). Such an association has been demonstrated for biliary alkaline phosphatase (Price et al, 1972); in this study of biliary alkaline phosphatase the effect of butanol extraction was to produce another isoenzyme with lower molecular weight and altered electrophoretic mobility, resulting from the removal of a phospholipid moiety from the biliary isoenzyme.…”

Serum gamma-glutamyltransferase isoenzymes in extrahepatic biliary obstruction.

“…Such an association has been demonstrated for alkaline phosphatase (Moss, 1962;Dunne et al, 1967;Jennings et al, 1970;Price and Sammons, 1974). In the case of yGT, it has been suggested that the biliary enzyme may be associated with lipid (Wenham et al, 1978a); furthermore, Beck (1978) suggested that a serum isoenzyme might be associated with a lipid component.…”

Value of differential thermostability, urea inhibition, and gel filtration of alkaline phosphatase in the identification of disease states.

“…Gel filtration studies were carried out as previously described (Dunne et al, 1967) by eluting 1 ml serum diluted with 2 ml of buffer through a 50 x 21 cm column of dextran gel (Sephadex G 200) containing 1 molar Tris NaCI buffer at pH 8.0. The rate of filtration was controlled by an LKB type 4912A Re-Cychrom infusion 45 pump.…”

“…Starch gel studies, while valuable in some hands, have at times produced conflicting results, particularly as regards the origin of normal serum phosphatase. Chiandussi, Greene, and Sherlock (1962) and Kowlessar, Haeffner, and Riley (1961), using starch gel electrophoresis, Dunne, Fennelly, and McGeeney (1967), using gel filtration, and Posen, Neale, and Clubb (1965), using heat inactivation, suggested an osseous origin for control serum enzyme, while Hodson, Latner, and Raine (1962) using similar techniques, and Yong (1967), using agar gel, concluded 'normal serum phosphatase is of hepatic origin'. Immunological studies of Boyer (1963) have shown definite cross reaction between skeletal and hepatic phosphatase, so that method is not suitable for application to clinical work in terms of differential diagnosis.…”