Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Studies on alkaline phosphatase. Transientstate and steady-state kinetics of Escherichia coli alkaline phosphatase

1. Reduction of a 19s immunoglobulin M with 3mm-mercaptoethanol or 0.05-0.5mm-dithiothreitol followed by alkylation gave sedimentation patterns indicating products compatible with structures consisting of one, two, three, four and five 7s sub-units. This supports the concept of a five-sub-unit structure for immunoglobulin M. 2. Reduction with 0.125mm-dithiothreitol or 20mm-cysteine produced 7s sub-units that could not be dissociated into chains in m-propionic acid. 3. By labelling (with iodo[2-(14)C]acetic acid) the thiol groups liberated during reduction with 0.125mm-dithiothreitol, it was possible to identify the tryptic peptides involved in the disulphide bridges that link the 7s sub-units together (inter-sub-unit bridges). 4. By further reducing and labelling (with iodo[2-(14)C]acetic acid) the 7s sub-units produced by 0.125mm-dithiothreitol, it was possible to identify tryptic peptides derived from intra-sub-unit bridges. 5. Sub-units produced by reduction with 20mm-cysteine proved to be unsuitable for distinguishing between inter-sub-unit bridges and intra-sub-unit bridges. 6. The possible arrangement of the interchain disulphide bridges was deduced.     

Leukocyte alkaline phosphatase in malignancies.

The leukocyte alklaine phosphatase (LAP) levels were determined in 183 patients with malignant diseases and 71 normal controls. The median LAP scores were 64 units (range 0 to 290) for the patients and 55 (range 2 to 158) for the controls, respectively, and no significant difference could be established. When analyzed according to primary malignancy, only in patients with Hodgkin's disease (n = 14) was the median value higher than normal (p less than 0.001). In patients with distant metastases (n = 48), higher LAP levels were demonstrated (M = 76, range 21 to 290) as compared to patients with no evidence of metastases (M = 53, range 0 to 229), (p less than 0.01). Thus, LAP activity has very limited value in the diagnosis of malignancies. Its elevation in the presence of malignant disease might, however, indicate metastases.     

Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.     

Bone alkaline phosphatase in Paget's disease.

The measurement of bone proteins and peptides and their derived products has been very useful in the diagnosis and management of patients with skeletal disease. This group of assays includes alkaline phosphatase (AP) and bone Gla protein (BGP). We here describe the comparison of a new immunoassay that is specific for bone alkaline phosphatase (BAP) to measurements of total alkaline phosphatase (TAP) and BGP in Paget's disease. In our studies, we demonstrated that BAP was increased in the serum of patients with Paget's disease. Comparisons with the other measurements revealed that BAP correlated better with total AP (r = 0.92) than with BGP (r = 0.51); the lowest correlation occurred between BGP and total AP (r = 0.26). In patients with liver disease, the BAP was indistinguishable from normal whereas the TAP was elevated. These studies indicate that BAP assesses different aspects of bone cell function than BGP in Paget's disease. This discordancy also exists between BGP and total serum AP activity. BAP measurements by immunoassay offer a novel method of assessing skeletal status. Thus, the information that measurement of different bone-specific proteins provides should be separately useful in assessing the skeleton for a variety of metabolic bone diseases.     

Monomeric alkaline phosphatase of Vibrio cholerae.

Alkaline phosphatase has been purified to homogeneity from two strains of Vibrio cholerae. The enzymes from both strains are single polypeptides of molecular weight 60,000. Both of the enzymes have pH optima around 8.0 and can act on a variety of organic phosphate esters, glucose-1-phosphate being the best substrate. The enzymes are unable to hydrolyze ATP and AMP. Although they have identical Km values, the two enzymes differ significantly in Vmax with p-nitrophenyl phosphate as substrate. The enzymes from the two strains also differ in their sensitivity to EDTA, Pi, and metal ions and activities of the apoenzymes. Ca2+ reactivated the apoenzymes most.