Mineralization of bone matrix may be influenced by the presence of specific, noncollagenous bone proteins. The quantitative influence of two bone-specific proteins--bone gamma-carboxyglutamic acid (Gla) protein and osteonectin--and other proteins that decreased the rate of crystal growth was measured by adding seed crystals of hydroxyapatite to a solution of CaCl2 and KH2PO4, pH 7.4 at 37 degrees C. The molar concentrations of proteins needed to inhibit the rate of crystal growth by 50% were as follows: osteonectin, 0.15 microM; bone Gla protein, 0.8 microM; prothrombin, 0.9 microM; prothrombin fragment 1, 1.0 microM; soybean trypsin inhibitor, 3 microM; prethrombin 1, 9 microM; cytochrome c, 30 microM. Calmodulin and parvalbumin were found to be less active than prothrombin fragment 1 and had no activity in the micromolar range. The combination of two inhibitors resulted in a mixture with an inhibitory activity that was the sum of the two inhibitors. Decarboxylation of bone Gla protein significantly reduced its inhibitory activity. These results indicate that the inhibitory activity of a protein does not correlate with Ca2+-binding affinity under these conditions, that the mixture of inhibitors has an additive effect, and that gamma-carboxyglutamic acid residues enhance the ability of a protein to inhibit hydroxyapatite-seeded crystal growth.

The Soret absorption maxima and extinction coefficients of the CO and NO complexes of horse myoglobin and (NMeIm)protoheme (NMeIm = 1-methylimidazole) have been determined. The partition coefficient N, equal to the ratio P1/2 (CO)/P1/2(NO), has been determined spectrophotometrically for horse myoglobin and (NMeIm)protoheme. P1/2-(NO) values calculated from the partition coefficients are 5.7 x 10(7) mmHg for (NMeIm)protheme and 1.1 x 10(6) mmHg for horse myoglobin. The ratio of P1/2(NO) values for protein and model is 1.9 which is similar to a value of 1.6 reported for the ratio of P1/2(O2) values. These values may be compared to a ratio of 15 for CO binding to protein and model complexes. This different ratio for CO provides further evidence for steric interaction of the bound CO with the protein based on a consideration of the preferred nonlinear geometry of Fe-NO and Fe-O2 and the linear geometry of Fe-CO.

No abstract

Routine methamphetamine testing identified a urine specimen with inconsistent screening and confirmation results. The methamphetamine RIA screening test (Diagnostic Products Corporation) indicated a borderline positive specimen, while the achiral confirmatory GC/MS result showed 4690 ng/mL of methamphetamine and 1895 ng/mL of amphetamine. Analysis of the specimen after derivatization with S(-)-N-trifluoroacetylprolyl chloride showed only the presence of 1-amphetamine and 1-methamphet-amine. It was later learned that the individual providing the specimen had been taking Selegiline. Selegiline, (-) propynylmethamphetamine, is a monoamine oxidase inhibitor used for the treatment of Parkinson's disease. It is sold under the trade name Eldepryl. Its major metabolites are 1-methamphetamine, 1-amphetamine and N-desmethylselegiline. Urine specimens from other Selegiline users were obtained and analyzed. A characteristic metabolic pattern was noted, exemplified by a ratio of 1-methamphetamine to 1-amphetamine of about 2.8. This is in contrast to what is observed in the urine of individuals who ingest pure 1-methamphetamine, such as with Vicks Inhaler, where the 1-methamphetamine to 1-amphetamine ratio in the urine is usually greater than 8. Caution is advised when interpreting methamphetamine results without using a chiral identification technique.

No abstract

Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J. A., Nesheim, M. E., Hibbard, L. S. & Mann, K. G.(1981) Proc. Nati. Acad. Sci. USA 78,[162][163][164][165][166] and Foster, W. B., Katzmann, J. A., Miller, R. S., Nesheim, M. E. & Mann, K. G. (1982) Thromb. Res. 28,[649][650][651][652][653][654][655][656][657][658][659][660][661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibodybound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 1251-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10-6) for anti-bone-Gla protein and (I50 -10-5) for antiosteonectin.

Absorption spectra were recorded for 5- and 6-coordinate model ferrous heme complexes of hindered and unhindered ligands in aqueous, and detergent solutions. Heme complexes exhibited differences in absorption maxima up to 6 nm which were correlated with the polarity of the heme environment. Increasing polarity of the solvent resulted in a general blue shift of absorption maxima of both deoxy- and (carbon monoxy)heme complexes. The differences in absorption maxima of heme complexes with different heme environments are offered as a possible explanation for some of the differences in absorption maxima among hemoproteins such as hemoglobin, myoglobin, and leghemoglobin.

The limits of linearity (LOL) and detection (LOD) are important factors in establishing the reliability of an analytical procedure for accurately assaying drug concentrations in urine specimens. Multiple analyses of analyte over an extended range of concentrations provide a measure of the ability of the analytical procedure to correctly identify known quantities of drug in a biofluid matrix. Each of the seven drugs of abuse gives linear analytical responses from concentrations at or near their LOD to concentrations several-fold higher than those generally encountered in the drug screening laboratory. The upper LOL exceeds the Department of Navy (DON) cutoff values by factors of approximately 2 to 160. The LOD varies from 0.4 to 5.0% of the DON cutoff value for each drug. The limit of quantitation (LOQ) is calculated as the LOD + 7 SD. The range for LOL is greater for drugs analyzed with deuterated internal standards compared with those using conventional internal standards. For THC acid, cocaine, PCP, and morphine, LOLs are 8 to 160-fold greater than the defined cutoff concentrations. For the other drugs, the LOL's are only 2 to 4-fold greater than the defined cutoff concentrations.