Deaminating enzyme labels for potentiometric enzyme immunoassay

Adenosine deaminase, asparaginase, and urease are examined as possible enzyme labels for immunoassays using potentiometric detection with the ammonia gas-sensing membrane electrode. Considerations of binding ability, retained activity, and stability reveal asparaginase to be the most effective enzyme label for immunoassay purposes. The utility of the potentiometric approach with this enzyme label is demonstrated via model hapten assays for dinitrophenyl groups and for cortisol.     

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Solid-phase immunoassay of dog neutrophil elastase

A sensitive and reliable method has been developed for the detection of dog neutrophil elastase using the amplified enzyme-linked immunosorbent assay. Purified neutrophil enzyme, inactivated by diisopropylfluorophosphate was adsorbed noncovalently to polystyrene tubes (11 × 55 mm) and immune rabbit serum was allowed to bind to antigen-sensitized tubes. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently lined to alkaline phosphatase, using p-nitrophenyl phosphate as the substrate. Increasing amounts of purified neutrophil enzyme or crude leukocyte extracts were quantitated by their ability to inhibit specific antibody uptake to polystyrene tubes. By this method, as little as 1 ng of enzyme/ml could be detected with a useful range of 5–100 ng of enzyme levels. Immunoreactive enzyme in a crude leukocyte extract was comparable to the quantily of enzyme as measured by proteolytic activity. The method described can be conveniently used to measure levels of immunoreactive enzymes in biological fluids of animals, with experimentally induced inflammatory conditions.     

Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.     

Beta-Galactosidase immunoassay for the measurement of cyclic AMP

The previously described method for phenotyping of alpha₁-antitrypsin (alpha₁-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.     

Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

The binding of Mn²⁺ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn²⁺ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?_b = 10.7 ± 2.0. The addition of chorismate to the Mn²⁺-enzyme complexes when predominantly the tight Mn²⁺ sites were occupied resulted in a large decrease in the observed enhancement (?_T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn²⁺ complexes caused large decreases in the water proton relaxation rate (?_T = 1.5) when tight or tight plus weaker Mn²⁺ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn²⁺ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn²⁺-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn²⁺ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn²⁺ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn²⁺-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn²⁺ or cause a large effect upon its environment.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

Rapid assay for in vitro degradation of luteinizing hormone releasing hormone

A radiochemical method for measuring luteinizing hormone releasing hormone (LHRH) degrading enzymatic activity in vitro was developed using LHRH labeled at the N-terminal 5-pyrrolidone-2-carboxylic acid (<Glu) residue. The intact labeled peptide is separated from the labeled fragments formed by cleavage by a cation-exchange batchwise procedure. The assay reflects the degradation of LHRH specifically in terms of inactivation of hormonal activity, is more rapid than a radioimmunoassay, is independent of LHRH concentration, and is not influenced by high protein concentrations. It can be used for studying the degradation of LHRH by subcellular fractions and enzymes. With this assay a highly active enzymatic degradation system was detected in the rat ovary, a recently discovered target organ for LHRH.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Stereochemical control of yeast reductions. 2. Quantitative treatment of the kinetics of competing enzyme systems for a single substrate

Quantitative expressions have been developed for systems such as yeast reductions where competing enzymes act on one substrate to yield two enantiomeric products. These expressions relate the observed stereochemical variables, the extent of conversion (C), the optical purity expressed as enantiomeric excess (ee), and the initial substrate concentration (A₀) to the kinetic parameters K_R and K_S (apparent Michaelis constants) and y ($\text{V}{}_{\mathrm{R}}\text{V}{}_{\mathrm{S}}$, the ratio of maximal velocities) of such competing enzymes. The expressions have been experimentally verified using a purified competing enzyme system of l- and d-lactic dehydrogenases. Furthermore, the enantioselective reduction of β-keto esters by intact yeast cells has been examined by means of this kinetic analysis.     

An enzyme-linked immunoassay for the cartilage proteoglycan

Proteoglycan was purified from a rat chondrosarcoma and antiserum prepared. An enzyme-linked immunoassay was designed using this serum. The assay detected rat and murine, but not chick, high-molecular-weight cartilage proteoglycan. It did not detect noncartilage proteoglycan nor the low-molecular-weight proteoglycans found in cartilage. As little as 100 ng/ml of rat cartilage proteoglycan could be detected.     

In vivo autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone $\text{in vivo}$ after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.     

Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: comparison with the bacterial collagenase from Achromobacter iophagus.

The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl-l-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds $\text{Leu}\text{15}\text{-}\text{Tyr}\text{16}\text{and}\text{Leu}\text{11}\text{Val}\text{12}$ as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.     

Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression

Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.     

Human liver acid phosphatases: Cysteine residues of the low-molecular-weight enzyme

The stoichiometry and the reactivity of the sulfhydryl groups of a human liver acid phosphatase have been studied. The smallest (M_r = 14,400) of the three molecular-weight forms of acid phosphatase from human liver, recently purified and characterized in our laboratory, was treated with various sulfhydryl group-specific reagents: p-hydroxymercuribenzoate, p-hydroxymercuriphenylsulfonate, fluorescein mercuriacetate, methyl methanethiosulfonate, p-nitrophenoxycarbonyl methyl disulfide, and thiosulfate. A total loss of enzymatic activity was obtained in each case. By spectrophotometric titration with 5,5′-dithiobis(2-nitrobenzoate) and p-hydroxymercuriphenylsulfonate it was shown that there are six free sulfhydryls per protein molecule, consistent with the amino acid analysis of this enzyme. The same number was deduced as a result of inactivation studies carried out with p-hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate. A total loss of activity was obtained at reagent to enzyme ratios of 6:1 in both cases. Similar results were obtained upon inactivation by p-nitrophenoxycarbonyl methyl disulfide, where the enzyme was found to possess only 10% residual activity at an inhibitor-to-enzyme ratio of 6:1. With fluorescein mercuriacetate as an inactivator, total loss of activity was found at a 2.5 times molar excess of this reagent over protein. Both the stoichiometry of inactivation and fluorescence titration experiments suggest that fluorescein mercuriacetate can function as a bifunctional sulfhydryl group reagent. The activity of a totally inactivated enzyme preparation obtained following reaction with excess of p-nitrophenoxycarbonyl methyl disulfide or with methyl methanethiolsulfonate could be almost completely restored upon treatment with dithiothreitol. These data are consistent with the interpretation that in each enzyme molecule, there are six free sulfhydryl groups of almost equal reactivity, at least one of which is essential for enzymatic activity.     

Progestin-induced enhancement of dexamethasone dissociation from glucocorticoid hormone receptors

Several groups have reported that progesterone accelerates the rate of steroid dissociation from the agonist site of the glucocorticoid receptor. It has been proposed that this enhancement reflects the binding of progestins to a second steroid-binding site. Since progestins are frequently antagonists of glucocorticoid hormone action, we decided to characterize this site more fully. In particular, in this study, we investigated whether the cytosolic preparations of four separate glucocorticoid target tissues from the same species all contained this second site and whether it was similar in each case. Cytosolic extracts of rat heart, liver, kidney, and pancreas were examined. In each case it was found that the rate at which prebound tritiated dexamethasone dissociated from the glucocorticoid receptor was faster in the presence of nonradioactive progesterone. The magnitude of this effect was essentially the same in each case. These results indicated that the second site was present in each preparation. To determine if the site was similar in each extract, we studied the steroid specificity of the enhancement of dissociation. This was determined by quantitating the degree to which each of a series of test steroids could cause augmentation of dissociation. Progesterone, R-5020, medroxyprogesterone, deoxycorticosterone, 17-OH-progesterone, and cortexolone were evaluated. The results for all four cytosolic preparations showed that either progesterone or R-5020 was the most potent steroid while both cortexolone and 17-OH-progesterone were essentially without effect. Medroxyprogesterone and deoxycorticosterone were usually of intermediate potency. These results suggest that the cytosolic extracts of all glucocorticoid target tissues have a similar second steroid-binding site which demonstrates a preference for progestins and that interaction with this site causes the glucocorticoid receptor to decrease the affinity with which it binds agonists.