Differential inhibition of the products of the human alkaline phosphatase loci

Abstract

1. Inhibition studies have been carried out on a series of ALPs derived from liver, bone, kidney, placenta and intestine, using L-phenylalanine, L-homoarginine, L-leucine, L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine as inhibitors. 2. No differences between liver, bone and kidney ALPs with any of the inhibitors were observed. 3. L-phenylalanine and L-homoarginine give a major degree of discrimination between liver/bone/kidney ALP on the one hand, and placental and intestinal ALPs on the other. L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine give a major degree of discrimination between placental ALP on the one hand, and intestinal ALP and liver/bone/kidney ALP on the other. L-leucine discriminates between the three classes, but to a lesser degree. Minor degrees of discrimination between placental and intestinal ALPs occur with L-phenylalanine and L-homoarginine and between intestinal and liver/bone/kidney ALPs with L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine. By using an appropriate combination of inhibitors the ALPs can be separated into three clearly distinct categories: placental, intestinal and liver/bone/kidney. 4. The six common placental ALP phenotypes as defined by electrophoresis show identical inhibition profiles with the series of inhibitors. The same profile was found for several rare electrophoretic variants. However, two rare electrophoretic variants (P-187 and P-92) each encountered once in a sample of 225 plancentae, showed significantly deviant inhibitions with the various inhibitors and also differed from each other. From the electrophoretic patterns, both of these rare phenotypes appear to be heterozygotes. P-187 probably corresponds to the so-called D-variant previously described. P-92 represents a new type of placental ALP variant with an aberrant inhibition profile. In both cases the particular rare allele concerned evidently alters the primary structure of the enzyme so that it has an altered electrophoretic mobility and also an altered sensitivity to inhibition for each of the different inhibitors. 5. Treatment of the various ALPs with neuraminidase to remove sialic acid residues does not affect their inhibition characteristics or activities.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue

“…The inhibitors used were levamisole (Sigma) and histidine (Sigma), which are inhibitors of tissue-non-speci¢c AP (TNAP), 15,16 and L-phenylalanyl-glycyl-glycine (PheGlyGly) (Sigma), which is an inhibitor of tissue-speci¢c AP. 17 The concentrations used were 2,50 and 20 mmol/L, respectively. The concentrations chosen were based on a previous study using the murine 3T3-L1 preadipocyte cell line.…”

Lipid accumulation and alkaline phosphatase activity in human preadipocytes isolated from different body fat depots

“…However, it should be noted that these inhibitors are sufficiently specific to have been routinely used to differentiate between the tissue-specific and tissue-non-specific isoenzymes of ALP. [2][3][4][5] In addition, and as noted previously, there is excellent evidence from other studies 9,10,28,30-32 of the involvement of ALP in intracellular lipid accumulation.…”

“…These inhibitors were added at the beginning of each differentiation period. The tissue-non-specific ALP inhibitors, histidine 2 and levamisole, 3 and the tissue-specific ALP inhibitor, PheGly-Gly, 4 were used at concentrations of 20 mmol/l, 2 mmol/l and 20 mmol/l, respectively. These doses were based on a previous study using the murine 3T3-L1 preadipocyte cell line.…”

“…2,3 The tripeptide, PheGly-Gly, is able to inhibit the tissue-specific ALPs, but not the tissue-non-specific isoform. 4 Serum levels of ALP have commonly been used for disease diagnosis, with the tissue-non-specific isoforms acting as markers of bone and liver pathology. 5 All adipose tissue depots are known to contain preadipocytes.…”

Electrophoresis of enzyme--monoclonal antibody complexes: studies of human placental alkaline phosphatase polymorphism.

“…They were the same as those used in our earlier report (4) and will be referred to as P3/1, Sp2/2, Sp2/ 3, etc., indicating the parental plasmacytoma cell line P3 (P3/ X63-Ag8) or Sp2 (Sp2/0-Agl4) and the serial number of the positive hybridoma. Placental extracts were prepared, assayed, typed electrophoretically, and, where required, treated with neuraminidase as described (7,8). ALPase-antibody complexes were prepared by using ALPase extract diluted to 0.1 international unit/ml with Tris-HCl, pH 7.4/0.15 M NaCl/0.02% sodium azide/0.05% Nonidet P-40 (British Drug House, Poole, England)/0.25% gelatin.…”