Evaluation of three immunoassays for detection of Chlamydia trachomatis in urine specimens from asymptomatic males.

The performances of three commercially available immunoassays (Chlamydiazyme/Antibody Blocking Assay [Abbott Diagnostics, Abbott Park, Ill.], IDEIA [Analytab Products, Plainview, N.Y.], and Microtrak EIA [Syva Co. Palo Alto, Calif.]) were evaluated for the detection of Chlamydia trachomatis in urine specimens from asymptomatic males. Assay results were compared with direct specimen immunofluorescence (DFA) analysis of urine sediment (Syva Microtrak; Syva Co.), which was chosen as the study confirmation assay. An overall Chlamydia prevalence of 7% (24 of 340) was found in our study population, with peak incidences occurring in the adolescent (8 of 93 specimens) and young adult (11 of 146 specimens) age groups. Sensitivity and specificity data among the Chlamydiazyme, IDEIA, and Microtrak enzyme immunoassays (EIAs) were determined to be 79.1 and 99%, 91.7 and 98%, and 95.8 and 99%, respectively. The Microtrak EIA and IDEIA products demonstrated sensitivities and specificities equal to or greater than those claimed for urine specimens. The diagnostic accuracies of these assays on asymptomatic subjects, along with the ease of this collection method, suggest a role for these products as screening tools. The sensitivity of the Chlamydiazyme assay was lower than that claimed previously in symptomatic patients, with 5 of 24 positive specimens demonstrating false-negative results. In those cases, centrifugation of the original immunoassay aliquot material and then DFA examination confirmed specimen positivity. Urine immunoassay screening in combination with DFA confirmation (which was chosen because it has antibody epitopic specificity different from that of the primary assay) provides a high degree of diagnostic precision. The use of noninvasive collection methods could result in greater testing compliance among asymptomatic males and, subsequently, could reduce the incidences of both symptomatic and silent chlamydial infections.

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b.

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

Binary affinity chromatography for the purification of glycogen phosphorylase.

A binary affinity chromatography medium was prepared and found to be useful for the purification and quantitative isolation of glycogen phosphorylase from rabbit skeletal muscle and liver. Glycogen is used as the binary ligand as it has affinity toward both the column matrix and the enzyme. Agarose beads derivatized with concanavalin A bound glycogen to the level of 35 mg/ml. The glycogen-impregnated beads were able to bind 9 mg/ml of phosphorylase a or b. The phosphorylase is tightly bound so that the column can be washed free of contaminants before quantitative elution of the phosphorylase by 2 M glucose, which releases the glycogen-phosphorylase complex. It appears that binary affinity chromatography may have general utility for the isolation and purification of enzymes and other specific binding agents.

Scanning tunneling microscopic images show a laminated structure for glycogen molecules.

Scanning tunneling microscopy (STM) has been used to examine glycogen molecules. Individual molecules were approximately ellipsoidal with dimensions in the 20- to 60-nm range. Images of the glycogen molecular surfaces have a laminar appearance. The layered features seen on the surfaces of the molecules suggest that glycogen may grow from one edge as a laminar structure to form an ellipsoid rather than originating at a central point with radial growth of the oligosaccharide chains to form a sphere. The results of these studies indicate that STM can be used to determine details of polysaccharide structures.

Viewing molecules with scanning tunneling microscopy and atomic force microscopy.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Scanning tunneling microscopy of the enzymes of muscle glycogenolysis.

Scanning tunneling microscopy (STM) has been used to examine the structures of the skeletal muscle enzymes phosphorylase and phosphorylase kinase. The interaction of these two proteins represents the last step in the process of signal transduction which results in muscle glycogen being converted into metabolic energy for use in muscle contraction. Phosphorylase b has a molecular weight of 97,000 and the dimer is seen by STM to have dimensions of 11 X 5.7 nm. Phosphorylase b has a tendency to form linear arrays of dimers on the graphite surface used as the support for STM imaging. Phosphorylase kinase is imaged as a butterfly-like object with lateral dimensions of 36 X 27 nm. The molecular thicknesses given by scanning tunneling microscopy for these two non-conducting molecules is significantly less than expected. The height measurement in STM is dependent not only on the surface topology of the object being imaged, but also on the electronic work function of the object compared to that of the graphite surface on which it lies. In addition to the individual proteins, a complex between phosphorylase and phosphorylase kinase has been observed by scanning tunneling microscopy.

Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy.

The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase.

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.

Inhibition of human lymphocyte cyclic nucleotide phosphodiesterases by the chlorinated adenosine analog DTA-35.

Human (E+)-T-lymphocytes have multiple cyclic nucleotide phosphodiesterase activities, some of which are markedly inhibited by the chlorinated adenosine derivative 9-(3',5'-dichloro-2',3',5'-trideoxy-beta-D-threo-pentofuranosyl)adenine (DTA-35).

Zero-order ultrasensitivity in the regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) is controlled by a cyclic phosphorylation-dephosphorylation process through the action of the interconverting enzymes, phosphorylase b kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and phosphorylase a phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17). In muscle tissue, the combined concentration of the activated (phospho-) form, phosphorylase a, and the nonactivated (dephospho-) form, phosphorylase b, is substantially greater than the Km of either of the interconverting enzymes for its phosphorylase substrate. It has been predicted that, under such a set of conditions, a sensitivity amplification will occur for phosphorylase regulation due to the zero-order ultrasensitivity effect [LaPorte, D. C. & Koshland, D. E., Jr. (1983) Nature (London) 305, 286-290]. The sensitivity amplification will enhance the responsiveness of the phosphorylase interconversion cycle to changes in the ratio of activities of the kinase to phosphatase. We have studied the cyclic interconversion process using purified muscle enzymes in steady-state reactions and found that there is an enhancement in the control sensitivity of the process due to the zero-order ultrasensitivity effect. The potential for the in vivo enhancement of sensitivity in glycogen degradation by this effect is discussed.

Inhibition by calmodulin of the cAMP-dependent protein kinase activation of phosphorylase kinase.

Calmodulin is shown to inhibit both the activation and phosphorylation of phosphorylase kinase by cAMP-dependent protein kinase. Maximal inhibition of both processes was approximately 66% at the highest calmodulin concentration tested (5.5 microM). It was found that the inhibition of phosphorylation was calcium-dependent, reversible by trifluoperazine, and specific for the beta subunit of phosphorylase kinase with no significant inhibition of phosphorylation of the alpha subunit. This inhibitory activity of calmodulin appears to be due to an interaction between calmodulin and the substrate, phosphorylase kinase. This finding implies either that the site of exogenous calmodulin interaction with phosphorylase kinase is at the beta subunit or that this interaction results in a conformational change of phosphorylase kinase that inhibits the interaction between cAMP-dependent protein kinase and the beta subunit of phosphorylase kinase. The beta subunit may contain a regulatory site that is recognized by either protein kinase or calmodulin. These findings further substantiate the role of the beta subunits in the activation of phosphorylase kinase and provide an additional example of substrate-directed control of phosphorylation.

Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci.

Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g.