Automated fluorometer/photometer system for homogeneous immunoassays.

A fully automated bench-top clinical analyzer (OPTIMATE TM; Ames/Gilford) performs homogeneous fluorescent immunoassays, colorimetric immunoassays, and determinations of routine blood analytes; drugs, enzymes, metabolites, specific proteins, and hormones in serum. Unique features include a combination fluorescence/absorbance aspirating thermocuvette, a photon-counting fluorometer/photometer, a multi-reagent distribution valve to dispense as many as three reagents plus buffer, and a user-replaceable programmable memory cartridge for software updates. We have evaluated the performance of OPTIMATE substrate-labeled fluorescent immunoassays for gentamicin, tobramycin, amikacin, theophylline, phenytoin, phenobarbital, primidone, carbamazepine, and quinidine with this automated system. A sample throughput of 92 samples per hour is achieved by reading fixed-point fluorescence results every 39 s after an initial 4-min reaction period. Precision studies indicate typical CVs of less than or equal to 6% for mid-range controls. Standard curves can be reused for as long as two weeks before recalibration. With clinical samples, results by the OPTIMATE procedure correlated well (r greater than or equal to 0.97) with those by a reference method.

Fluorescence studies of the interaction of calmodulin with myosin light chain kinase.

The interaction of calmodulin with myosin light chain kinase produces an approximately 30% increase in myosin light chain kinase tryptophan fluorescence. This represents the first report of calmodulin-induced structural changes in a protein which it activates. We fund that the calmodulin-myosin light chain kinase interaction is: 1) dependent on [Ca2+] (half-maximal binding at pCa 6.2) and essentially independent of [Mg2+], 2) occurs before saturation of all four reported Ca2+-specific sites on calmodulin. 3) saturates with 1 mol of calmodulin bound per mol of kinase with an apparent affinity of approximately 2.0 X 10(7) M-1, 4) is specific for calmodulin over troponin-C, 5) is directly related to the activation of myosin light chain kinase for phosphorylation of myosin light chain. Fluorescence stopped flow studies of these calmodulin-induced fluorescence changes in myosin light chain kinase indicate that Ca2+ binding to calmodulin occurs very rapidly and is not rate-limiting while the calmodulin-induced fluorescence increase in myosin light chain kinase occurs as a biphasic process with rates of approximately 65 s-1 and 6 s-1. The fluorescence increase produced by calmodulin binding to myosin light chain kinase is completely reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at a rate of approximately 2 s-1.

Interaction of calmodulin with skeletal muscle myosin light chain kinase.

Studies on myosin light chain kinase isolated from rabbit skeletal muscle show that the enzyme has a molecular weight of 80,000--84,000 with a sedimentation coefficient of 3.2 S and an apparent Stokes radius of 53 A. Gel filtration chromatography with a 3H-labeled calmodulin using a Hummel--Dryer technique shows that the enzyme will bind 1 mol of calmodulin per mol of enzyme, with an affinity of (1.9 +/- 0.5) x 10(7) M-1 in the absence of substrate. The calmodulin dependence of enzyme activation at limiting Mg2+ and light chain concentrations confirms this observation. The calcium dependence of activation of the enzyme--calmodulin complex is characterized by a Hill coefficient of 2.5, with half-activation occurring at 6.6 x 10(-7) M Ca2+. The amino acid composition shows a high percentage (9.1%) of proline, which may account for the large apparent Stokes radius and no clear resemblance to other skeletal muscle proteins. A comparison of the amino acid composition with that from turkey gizzard shows some resemblance.

Positive cooperative binding of calcium to bovine brain calmodulin.

Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.

Calcineurin: a calcium- and calmodulin-binding protein of the nervous system.

The inhibitory protein that binds calmodulin and thus prevents activation of several Ca2+-dependent enzymes by calmodulin is shown to also bind four Ca2+ per mol of protein with high affinity (Kd less than or equal to 10(-6) M). On the basis of its Ca2+- binding properties and its localization to nervous tissue, the inhibitory protein is now called "calcineurin." Calcineurin is composed of two subunits: calcineurin A (61,000 Mr) which interacts with calmodulin in a Ca2+-dependent fashion, and calcineurin B (15,000 Mr) which binds Ca2+. The interaction of calcineurin A with calcineurin B is independent of Ca2+ or Mg2+. The dual interaction of calcineurin A with two different Ca2+-binding components and the high affinity of calcineurin for Ca2+ suggest a possible role for calcineurin in the regulation of free Ca2+ concentrations in the nervous system. Calcineurin may thereby modulate the release and action of neurotransmitters.

An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart.

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.