The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.

The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.     

Decline in serum antibodies to methyltetrahydrophthalic anhydride after cessation of exposure.Implications for use as a biomarker

T he association between exposure intensity and serum levels of immunoglobulins E and G against low molecular weight compounds was evaluated. The decay of levels of specific IgE and IgG antibodies was studied after cessation of exposure in workers exposed to the inhalant allergen methyltetrahydrophthalic anhydride in a plant using epoxy resins. Sera have been collected in workers for 18-84 (mean value 54) months after cessation of exposure. Specific IgE and IgG was assessed by RAST and ELISA, respectively. The mean of individual half-times for IgE ( N = 10) and IgG ( N = 8) was 0 9 (range 0 1-1 8) and 0 4 (range 0 2-0 6) years, respectively, after total avoidance of exposure. Corresponding decreases of IgE and IgG were also observed after reduction, but not total elimination, of exposure. No correlation was seen between biologic halftimes of specific IgE and total IgE, atopy, smoking habits or gender. The results indicate that the levels of specific antibodies in sensitized individuals reflect long term exposure, and may persist for years after the end of exposure.     

The use of coated paramagnetic particles as a physical label in a magneto-immunoassay

An ideal label for use in an immunoassay would require no further chemical or electromagnetic stimulation prior to its detection and would be free from interference from the sample matrix. Micron sized paramagnetic particles are able to perturb magnetic fields. This perturbation can be directly detected using a suitable electronic device and is independent of the sample matrix. In this study coated paramagnetic particles were used as a physical label in a non-competitive solid phase ‘sandwich’ assay for the detection of human transferrin. The transferrin acted as a ‘biological bridge’ allowing a dose dependant immobilization of the paramagnetic particles to a polyethylene terephthalate solid phase. Quantitation of the paramagnetic label was achieved using an electronic detection system allowing a linear dose response with a femtomolar detection limit (260 fmol).     

Polyclonal antibodies to chlorosome proteins as probes for green sulfur bacteria.

We found that polyclonal antibodies raised against chlorosome polypeptides from green sulfur bacteria reacted to Chlorobium tepidum, Chlorobium limicola, and Chlorobium phaeobacteroides but not to Chloroflexus aurantiacus. These antibodies successfully labeled only green sulfur species in marine microbial mat samples. Our results suggest that these antibodies may be useful as immunohistochemical probes.