Evidence of alkaline phosphatase interference in a zidovudine radioimmunoassay.

Phosphorylated zidovudine (ZDV) concentrations may provide a link between drug exposure and clinical efficacy since these would include the active, intracellular form of the drug, ZDV triphosphate. Many groups are investigating the optimal methodology that can be used to accomplish this goal. The initial purpose of the present studies was to examine the effect of the inclusion of cell wash steps on the quantitation of intracellular ZDV. Ten milliliters of whole blood collected from healthy volunteers was spiked with increasing ZDV concentrations (0.187, 0.375, 1.87, and 3.75 microM), allowed to equilibrate at room temperature for 1 h, and separated into whole-blood components by a density gradient procedure. A mononuclear cell pellet was obtained, reconstituted with 2 ml of phosphate-buffered saline (PBS), and split into two aliquots, one of which was not washed at all and the other of which was washed four times with 1 ml of PBS. All samples were analyzed by ZDV radioimmunoassay (RIA) after a 1:1 dilution with either 1 mg of alkaline phosphatase (type 1-S; Sigma) per ml or PBS. Parent ZDV was measured in those samples which were not treated with the enzyme, while total ZDV was measured in those samples which were exposed to alkaline phosphatase (21 degrees C for 1 h). The result of the difference between the two samples is total phosphorylated ZDV. During the experiment, evidence of alkaline phosphatase interference with the RIA became apparent, confusing interpretation of intracellular ZDV concentrations. This evidence was based on three sets of data. First, wash samples showed increases in ZDV concentrations of as great as 0.127 microgramM after exposure to alkaline phosphatase, even though on microscopic inspection the wash samples were acellular. Second, the sum of total ZDV recovered from the four wash samples plus the washed cell pellet was as much as 14-fold greater than the total ZDV measured in the unwashed cell pellet. Theoretically, at least, these two entities should be equal. Finally, control samples of alkaline phosphatase in PBS (0.5 mg/ml) run directly through the assay measured false ZDV levels ranging from 0.002 to 0.075 microgramM (0.6 to 20 ng/ml). Alkaline phosphatase is frequently used to measure phosphorylated anabolites of ZDV in peripheral blood mononuclear cells. These data show that the particular form of alkaline phosphatase used may interfere with the ZDV RIA and may confuse the interpretation of phosphorylated anabolite concentrations of ZDV.

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain. II. Enzymatic characteristics and regulation of activities by phosphorylation and dephosphorylation.

In addition to physical properties (DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13460-13465), enzymatic and regulatory characteristics indicate that calmodulin (CaM) kinase Ia and CaM kinase Ib are distinct entities. The Km values for ATP and site 1 peptide were similar between the two kinases, however, CaM kinase Ib is approximately 20-fold more sensitive to CaM than is CaM kinase Ia. The kinases also displayed differential sensitivities to divalent metal ions. For both kinases, site 1 peptide, synapsin I, and syntide-2 were highly preferred substrates relative to others tested. A 72-kDa protein from a heat-treated extract of rat pancreas was phosphorylated by CaM kinase Ib but not by CaM kinase Ia. CaM kinase Ia activity displayed a pronounced lag in its time course suggesting enzyme activation over time. Preincubation of CaM kinase Ia in the combined presence of Ca(2+)-CaM and MgATP led to a time-dependent increase in its site 1 peptide kinase activity of up to 15-fold. The extent of activation of CaM kinase Ia correlated with the extent of autophosphorylation. The enzyme retained full Ca(2+)-CaM dependence in the activated state which was rapidly reversible by treatment with protein phosphatase 2A catalytic subunit. Thus, the activation of CaM kinase Ia is a result of its Ca(2+)-CaM-dependent autophosphorylation. CaM kinase Ib was not activated by preincubation under autophosphorylating conditions yet lost approximately 90% of its activity toward either an exogenous substrate (site 1 peptide) or itself (autophosphorylation) after incubation with protein phosphatase 2A catalytic subunit. The deactivated state was not reversed by subsequent incubations under autophosphorylating conditions. Thus, CaM kinase Ib activity is dependent upon phosphorylation by a regulating kinase(s) which is resolved from CaM kinase Ib during purification of the latter.