Alkaline Phosphatase 333
coefficient of BAP, S°20,w, is 6.0 at pH 8.0 (11) and its isoelectric point
pH 4.5 (12).
Isozymes have been detected (13)--normally three, but more have
been reported. Active dimers can be formed from monomers derived
from different sources (13).
The dimeric form of the enzyme contains two atoms of zinc, which are
required for activity. The dimer dissociates to monomers at pH <3.0
with release of zinc ions, and chelating agents can also remove the zinc
atoms, but without the formation of monomers in this case. Some
workers report four atoms of zinc per dimer, only two being required
for activity (14), and inactivation of the enzyme by metal chelators is
biphasic. There is one active site per dimer at low substrate concentra-
tions (<0.1 mM) (15) and two at higher concentrations (>1 mM) (16).
2.2. Calf lntestinal Alkaline Phosphatase (CLAP)
CIAP is a glycoprotein with a mol mass of 100,000-140,000 Da
(17,18), comprising two identical or similar subunits and containing
four atoms of zinc per dimer (18,19). Isozymes have been found of
placental alkaline phosphatase, but not ClAP (1). The enzyme has an
isoelectric point of 5.7 independent of temperature (in the range 15-
25°C) and ionic strength (0.02-0.5) (20).
- Enzymic Reaction
3.1. Substrate
Substrates for alkaline phosphatase are varied ranging from the
phosphate esters of primary and secondary alcohols, sugar alcohols,
and phenols to nucleotides and nucleic acids. This nonspecificity is
reflected in the similar rates of hydrolysis of a wide range of substrates
(21). Trans-phosphorylations also occur, often to alcohol moieties in
the buffer other than water, such as Tris or ethanolamine.
Of interest to the molecular biologist is the action on the 5'-phos-
phate group of single- or double-stranded DNA or RNA that yields a
5'-hydroxyl group (22,23). The enzyme exhibits further activity on RNA
and can hydrolyze 5'-di- and triphosphate groups (24), and 3'-phos-
phate groups (22). It can also cleave 2-', 3'-, and 5'-phosphates of
mononucleosides (21). Only phosphate monoesters are susceptible
and diesters are not reactive, although some alkaline phosphatase
preparations will also act as pyrophosphatases. This activity can be pre-