Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.     

Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques.

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.     

An immunoenzymatic method for improving the sensitivity of antigen measurement by electroimmunodiffusion techniques. Application to the quantification of human alpha-fetoprotein.

A double antibody technique of electroimmunodiffusion, which uses glucose oxidase-labelled sheep antibodies to rabbit immunoglobulins as second antibody, is described. Primary antigen-antibody reaction is carried out with a rabbit antiserum by electroimmundiffusion. The glucose oxidase-labelled immunoreagent, being of general application, can serve for the quantification of different antigens and is here used for measurement of low levels of human alpha-fetoprotein. Reproducible results in the range of 50-800 ng/ml were obtained with a variation coefficient of 5 to 10%.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

Application of the theory of enzyme subunit interactions to ATP-hydrolyzing enzymes. The case of Na,K-ATPase.

The theory developed by T. L. Hill (1977, Proc. Natl. Acad. Sci. USA, 74:3632-3636) for enzyme interactions is applied to a dimeric enzyme, the subunits of which may each exist in three distinct states (as in a uni-bi kinetic mechanism). It is shown that when simultaneous binding of substrate to both subunits is excluded, the complex kinetic mechanism of the dimer reduces to a simpler scheme with two distinct, but analogous, cycles that are in principle separately observable in kinetic experiments. Because of the intersubunit interactions, which are explicitly taken into account, the two cycles have different Michaelis constants and maximal velocities. The model exhibits negative cooperativity and enhanced reactivity, relative to a monomeric enzyme. The theory is applied to Na,K-ATPase for which a complete, bicyclic, kinetic mechanism and rate constants are available. When taken together with other evidence, structural as well as functional, the striking similarity of the observed kinetics with that developed for a dimeric enzyme strongly suggests that the functional unit of Na,K-ATPase is a dimer. The free energy differences (calculated from the known rate constants) between intermediates are 6-16 kJ/mol, comparable, for example, to the free energy associated with the formation of a base pair in a nucleic acid double helix. The possible relevance of these results for other ATPases is briefly discussed.     

Comparative usefulness of cytology and peroxidase activity in conjunction with carcinoembryonic antigen levels for the diagnosis of mammary cancer

Diagnosis of cancer via measurement of carcinoembryonic antigen (CEA) levels has been unreliable in early neoplastic stages. In order to improve diagnostic reliability, other cytological parameters were examined with CEA. Fifty specimens of effusion fluid were obtained from 40 hospitalized patients and the levels of CEA determined by radioimmunoassay in conjunction with application of an immunoperoxidase procedure. Simultaneous morphologic assessment was performed without knowledge of the immunoassay findings. In 8 documented cases of mammary cancer, all effusion fluid specimens had CEA levels of 16-1074 ng/ml, 7 cases had morphologically positive cells, but only 3 had a peroxidase positive reaction. Except for one case of ovarian papillary adenocarcinoma, the remaining patients were cancer free, had CEA levels of less than 15 ng/ml and only 2 cases (including the ovarian tumor patient) gave positive peroxidase responses. The presence of mammary metastatic duct carcinoma correlated 88% with CEA measurements but peroxidase response was not diagnostically helpful.     

Cloning part of the region encoding biosynthetic enzymes for surface antigen (O-antigen) of Salmonella typhimurium

Summary The rfb gene cluster of Salmonella typhimurium encodes the enzymes required for the biosynthesis of the O-Antigen. A part of it has been cloned in plasmid vectors pBR322 and pUC9 using an adjacent, previously cloned, part of the his operon (Barnes 1981) as a molecular probe for the first clone. A detailed restriction enzyme map of 7.57 kb of rfb DNA is presented and the approximate locations of two of the genes, rfbK and rfbM have been defined.     

Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmol/assay and LLQ = 30 pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09 nmol hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmol of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.     

Impact of preoperative CA 15-3 levels in operable breast cancer. Comparison with tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA)

CA 15-3, TPA and CEA were assayed before surgery in 60 patients with breast cancer. A significant association was found between preoperative CA 15-3 levels and some of the most important prognostic factors in breast cancer, such as lymph node status and tumor size. No similar association was discovered for CEA and TPA. Preoperative CA 15-3 levels were also significantly associated with early recurrences of the disease, thus adding useful information to prognosis especially in N+ patients.     

Free radical formation in the oxidation of malondialdehyde and acetylacetone by peroxidase enzymes.

Malondialdehyde, a product of lipid peroxidation, and acetylacetone undergo one-electron oxidation by peroxidase enzymes to form free radical metabolites, which were detected with ESR using the spin-trapping technique. The structures of the radical adducts were assigned using isotope substitution. With both malondialdehyde and acetylacetone and the enzymes myeloperoxidase and chloroperoxidase, carbon-centered radicals were detected. With horseradish peroxidase, a carbon-centered radical was initially trapped and then disappeared with the concomitant appearance of an iminoxyl radical.     

Application of the synthetic method to other amines.

Taurine and 2-aminoethylphosphonic acid were synthesized by the method of the main paper [Geoghegan & Dixon (1989) Biochem. J. 260, 295-296], i.e. by treating the corresponding halo compound with 2-aminoethanol and then with periodate.     

Action of calcium ions on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase and other enzymes of the Calvin cycle.

Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase is powerfully inhibited by Ca2+ non-competitively with respect to its substrate, fructose 1,6-bisphosphate. 500 microM-Ca2+ causes virtually complete inhibition and the Ki is 40 microM. Severe inhibition of sedoheptulose bisphosphatase is also caused by Ca2+. A role for Ca2+ in regulation of the Calvin cycle in spinach chloroplasts is proposed.     

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.     

Application of fluorescamine to the study of protein-DNA interactions.

The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes.     

Novel spin-labels for the study of lipid-protein interactions. Application to (Na+, K+)-ATPase membranes

The interactions of a series of spin-labeled fatty acids, in which the nitroxide ring is incorporated in different ways as an integral part of the hydrocarbon chain, with the (Na+,K+)-ATPase in membranes from Squalus acanthias, have been studied by electron spin resonance spectroscopy. The fatty acids are 2,4-, 2,5-, and 3,2-substituents of 2,2,5,5-tetramethylpyrrolidine-N-oxyl and belong to the class of minimal perturbation nitroxide probes. For all five fatty acid labels, a motionally restricted lipid component was observed in the ESR spectra of (Na+,K+)-ATPase membranes, in addition to the fluid component, which was found in the spectra of the extracted membrane lipids. The pH dependence of the motionally restricted spin-label population indicated a sensitivity in the selectivity of the lipid-protein interaction to the protonation state of the fatty acid. These results agree with those found previously for the conventional oxazolidine (doxyl) fatty acid and phospholipid spin-label derivatives [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] and indicate that the motion of the lipid chains is significantly hindered by interaction with the protein, irrespective of the nature of the spin-label group.