Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity

Abstract

1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.

Phosphorylation by Alkaline Phosphatase: Immobilization and Synthetic Potential

“…With 250 mM PP i , the reaction is also faster at pH 8 but the reaction is very slow and reaches a steady level with no further hydrolysis (data not reported). This suggests a possible inhibition by the substrate PP i , as several authors already reported (Butterworth, 1968;Fernley & Walker, 1967;Morton, 1955;Nayudu & Miles, 1969). This is in contrast to the acid phosphatases PhoN-Sf and PhoN-Se, which completely consume PP i under similar conditions and which are not inhibited by PP i (Tanaka et al, 2003;van Herk et al, 2005).…”

“…Furthermore the AP is commercially available in contrast to the bacterial acid phosphatases. As shown by us PP i did not inhibit the alkaline phosphatase from bovine intestine, but the product of the reaction, P i , had a strong inhibitory effect on the activity of AP as found by many authors (Butterworth, 1968;Fernley & Walker, 1967; www.ccsenet.org/ijc Morton, 1955;Nayudu & Miles, 1969). Already at 10 mM of P i the hydrolysis of PP i was inhibited for 10 % and at 25 mM P i 40 % inhibition was found.…”

Alkaline phosphatase activity associated to a calcium binding glycoprotein from calf scapula cartilage

“…A good inhibition is also demon~ strated by inorganic phosphate, one of the reaction products, hs reported in literature for this enzyme [18]. L-Phenylalanine is considered an organo-specific and stereo-specific inhibitor of the intestine alkaline phosphatase [19].…”

The importance of dissolved organic phosphorus to phosphorus uptake by limnetic plankton

“…AP is a nonspecific PMEase that recognizes terminal phosphates of many PME and even short polyphosphate chains (McComb et al 1979), whereas 5PN recognizes the carbon moiety of 5'-nucleotides (Bengis-Garber and Kushner 198 1). ATP often has been used as substrate in studies with purified AP enzymes of E. coli and marine bacteria (Heppel et al 1962;Fernley and Walker 1967;Friedberg and Avigad 1967;Kobori and Taga 1980), and [32P]ATP has been used to measure P hydrolytic rates by both AP (Hulett-Cowling and Campbell 197 1; Reid and Wilson 197 1) and 5PN enzymes (Bengis-Garber and Kushner 198 1,1982;Ammerman and Azam 1985). Currently, nucleotides are the only convenient choices for 32P-labeled PME studies, as other 32P-labeled PME are not commercially available.…”