Donald W. Moss

Association of alkaline phosphatase with an autoantigen recognised by circulating anti-neutrophil antibodies in systemic vasculitis

A solid phase radioimmunoassay has been developed to detect circulating autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis. After fractionation of these antigens by size, with gel filtration high performance liquid chromatography, sera from patients with clinically different forms of systemic vasculitis, Wegeners granulomatosis (WG) and microscopic polyarteritis (MP), showed contrasting specificities of binding. WG sera bound to 100, 6.2, and 1.8 kD components, whereas MP sera bound only to the 100 kD component, allowing immunological distinction between the syndromes. The 100 kD component recognised by both WG and MP sera also showed alkaline phosphatase activity. Further evidence for this association was obtained by direct binding experiments between systemic vasculitis sera and calf-intestinal or human neutrophil alkaline phosphatase and by the cross-reactivity of W8, a monoclonal antibody raised to a neutrophil cytoplasmic autoantigen, with various preparations of the enzyme.

Inhibition of osteoclastic acid phosphatase abolishes bone resorption.

Osteoclastic acid phosphatase is a member of a widely-distributed class of iron-containing proteins with acid phosphatase activity. Antibodies raised against one member of this class cross-react with other members from the same or different species, but not with acid phosphatase isoenzymes of different types. When antibodies to one such protein, porcine uteroferrin, are added to medium in which rat osteoclasts are incubated on devitalised cortical bone, both bone resorption and acid phosphatase activity are markedly inhibited. Furthermore, addition of molybdate (an inhibitor of this class of acid phosphatases) also inhibits both bone resorption and enzyme activity. These observations strongly suggest a functional role for osteoclastic acid phosphatase in bone resorption.

Diagnostic aspects of alkaline phosphatase and its isoenzymes

The changes in serum alkaline phosphatase that are of main diagnostic importance result from increased entry of enzyme into the circulation. This results from increased osteoblastic activity in bone disease, and increased synthesis of alkaline phosphatase by hepatocytes in hepatobiliary disease. The liver and bone forms of alkaline phosphatase are differently-glycosylated forms of a single gene product. The main value of their specific estimation is found in patients in whom bone and liver diseases co-exist, for example, as a result of cancer. Abnormal expression of genetically-distinct alkaline phosphatase isoenzymes is valuable in monitoring cancers, particularly germ-cell tumors. These isoenzymes include Regan and Nagao isoenzymes, which correspond respectively to normal placental and placental-like alkaline phosphatases, and the Kasahara isoenzyme which appears to result from re-expression of a fetal intestinal alkaline phosphatase gene.

Multiple forms of acid and alkaline phosphatases: Genetics, expression and tissue-specific modification

It is now rather more than 25 years since the idea that, within a single organism, the same chemical reaction may be catalysed by several structurally distinct enzyme proteins became accepted as a major generalization in biochemistry. What was at first merely a collection of observations on the phenomenon of enzyme heterogeneity quickly became rationalized and codified into a scheme which recognizes the existence of true isoenzymes, encoded by distinct structural genes (including any heteropolymers which may be formed from unlike subunits in the case of oligomeric enzymes), and which distinguishes these true isoenzymes from multiple enzyme forms which may arise by processes of co-translational or post-translational modification. The different structural genes which encode the molecules (or subunits) of true isoenzymes may be due to existence of multiple genetic loci, common to all members of the species, or to the existence of allelic variation at enzyme-determining loci. Many of the practical clinical applications of multiple forms of enzymes, notably their use in sensitive and specific diagnostic tests, are independent of a knowledge of their molecular nature and origins. Nevertheless, such knowledge is valuable not only for the insights that it provides into normal and pathological metabolism, but also in the design of appropriate analytical methods. Some generalizations can be made with greater or lesser degrees of certainty about isoenzymes determined by multiple gene loci: for example, they differ in catalytic properties such as substrateaffinity or response to inhibitors, presumably reflecting their differently-evolved metabolic roles; they can be expected to be antigenically distinct, though monoclonal antibodies may be required to demonstrate this, and they are likely to differ in stability. The probable differences between ‘allelozymes’, i.e. isoenzymes that originate from the existence of allelic genes at a given locus, are much less predictable, deriving as they do from individual random mutations; nevertheless, monoclonal antibodies are potentially capable of distinguishing all allelozymic forms of an enzyme molecule.

Activities of bone and liver alkaline phosphatases in serum in health and disease

A heat-inactivation method for determining absolute activities of liver and bone alkaline phosphatases in serum has been applied extensively in routine diagnosis. Values for each isoenzyme in healthy individuals of different ages are reported together with results obtained in various diseases. Data from normal subjects show that bone alkaline phosphatase contributes about half the total alkaline phosphatase activity in adults. Liver phosphatase shows a slight increase with age. The method is also able to detect reliably the presence of carcinoplacental isoenzymes.

Clinical and Biological Aspects of Acid Phosphatase

The identity and genetic origins of the nonspecific orthophosphate monoesterases with an acid pH optimum--the acid phosphatases--are now becoming clear. They form a family of genetically distinct isoenzymes, many of which show significant posttranslational modification. Four true isoenzymes exist. The erythrocytic and lysosomal forms show widespread distribution and are expressed in most cells; in contrast, the prostatic and macrophagic forms have a more limited expression. The erythrocytic and macrophagic forms are distinguished from the others in resisting inhibition by dextrorotatory tartrate. The prostatic form has long been used as a marker for prostatic cancer and the macrophagic forms have been linked with miscellaneous disorders, notably increased osteolysis, Gauchers disease of spleen, and hairy cell leukemia, whereas the normal levels of intravesical lysosomal acid phosphatase in I cell disease pointed the way toward the mechanisms underlying its intracellular processing.

Physicochemical and pathophysiological factors in the release of membrane-bound alkaline phosphatase from cells☆

Alkaline phosphatase is bound to cell membranes by a glycan phosphatidylinositol anchoring domain. The structure of this domain and ways in which it may be cleaved by chemical and enzymatic means provide a basis for understanding the solubilization of alkaline phosphatase from tissues in vitro and in vivo and the generation of isoforms.

Release of membrane-bound enzymes from cells and the generation of isoforms

Enzymes bound to the surfaces of cells may be retained by a hydrophobic amino acid sequence (e.g. gamma-glutamyltransferase) or by a specific glycan phosphatidylinositol (GPI) anchor (e.g. alkaline phosphatase). In either case the attachment is by means of non-covalent hydrophobic interactions between the anchoring domain of the enzyme and lipid components of the cell membrane. Enzyme molecules released into the plasma or bile, complete with their hydrophobic domains, can undergo aggregation and complexation to give rise to high molecular weight isoforms of gamma-glutamyltransferase or alkaline phosphatase. However, the GPI domain of alkaline phosphatase can be degraded by an inositol-specific phospholipase in plasma, but not in bile, with production of the hydrophobic, non-aggregating isoform of alkaline phosphatase that predominates in plasma.

Modification of Alkaline Phosphatases by Treatment with Glycosidases

The tissue-specific variants of alkaline phosphatase that are characteristic of human liver and bone are believed to possess identical protein cores; nevertheless, they differ in certain properties such as electrophoretic mobility and stability to heat. Their electrophoretic mobilities are modified by digestion with various glycosidases. Furthermore, the difference in heat stability between them is reduced by treatment with a glycosidase preparation from Trichomonas foetalis. These results are consistent with the view that these enzyme variants differ only in their carbohydrate moieties.

Malabsorption and macroamylasemia response to gluten withdrawal

A 36 year old woman presented with malabsorption and macroamylasemia. The macroamylase was characterized and shown to be a complex of pancreatic amylase and immunoglobulin A(IgA). The patient had the clinical and histologic features of adult celiac disease, and responded to a gluten-free diet. The macroamylase complex disappeared from the serum after gluten withdrawal, a hitherto unreported finding in the syndrome of malabsorption and hyperamylasemia.