A rapid,sensitive assay for starch phosphorylase and ADPglucose pyrophosphorylase

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

A radiochemical assay for N5-formyltetrahydrofolic acid (citrovorum factor) in serum and urine.

N⁵-Methyltetrahydrofolate, but not N⁵-formyltetrahydrofolate, can be measured in biological fluids by ligand-binding radioassay. Therefore, in order to measure N⁵-formyltetrahydrofolate by radioassay, it is chemically converted to N⁵-methyltetrahydrofolate by acidification followed by reduction with borohydride. By this method, 70–113% of N⁵-formyltetrahydrofolate added to serum and urine was recovered. The plasma clearance of the mixture of diastereoisomer of N⁵-formyltetrahydrofolate (Leucovorin) following intravenous administration to two normal subjects was rapid for the first 30 min, but then plateaued and cleared very slowly over the next 90 min, most probably because of the accumulation of the inactive isomer which was slowly excreted in the urine during this time period.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Specificity of the dopamine sensitive adenylate cyclase for antipsychotic antagonists

Chlorpromazine, haloperidol and clozapine are approximately equipotent in antagonizing dopamine sensitive adenylate cyclase activity in homogenates of rat brain striatum, in contrast to the differences in clinical antipsychotic potencies reported by others. The antagonism appeared to occur at a structurally specific dopamine site, as inhibition by a series of chlorpromazine analogues of similar hydrophobicity exhibited a structural specificity similar to that found for their neuroleptic and cataleptic activities. Sulpiride, a dopamine antagonist with antipsychotic activity, and metoclopramide, a structurally related central dopamine antagonist, failed to inhibit the dopamine sensitive adenylate cyclase. Pre-treatment of rats with haloperidol (3 mg/kg per day) for 6 or 28 days did not induce a supersensitive response of the adenylate cyclase to stimulation by dopamine or apomorphine or inhibition by clozapine. It was concluded that the dopamine sensitive adenylate cyclase may not be the site of action of all anti-psychotic agents.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine,epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three ³H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.     

An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).     

The utility of the microsomal 4-chlorobiphenyl hydroxylase enzyme assay in distinguishing between phenobarbitone- and 3-methylcholanthrene-induced microsomal monooxygenases

4-Chlorobiphenyl was used as a substrate for the in vitro determination of rat hepatic microsomal, cytochrome P-450-dependent monooxygenase activity. The 4-chlorobiphenyl hydroxylase assay was tested for its ability to distinguish between a variety of phenobarbitone- and 3-methylcholanthrene-type inducers. Two radiometric procedures were employed to investigate the metabolism of 4-chlorobiphenyl. First, the metabolite profile of 4-chlorobiphenyl was analyzed by radio-thin-layer chromatography. This procedure permitted an assessment of the effects of microsomal enzyme inducers on both the qualitative and quantitative aspects of 4-chlorobiphenyl metabolism. Second, the rate of 4-chlorobiphenyl metabolism was determined by a differential extraction procedure which separated unreacted starting material (hexane phase) from metabolites (base phase). This procedure provided a rapid measurement of the overall activity of 4-chlorobiphenyl hydroxylase. Irrespective of the animal pretreatment, the metabolite profile of 4-chlorobiphenyl was dominated by 4′-chloro-4-biphenyl. Unlike the qualitative aspects, the quantitative aspects of 4-chlorobiphenyl metabolism were markedly influenced by animal pretreatment. Specifically, 3-methylcholanthrene-type inducers (3-methylcholanthrene and 3,3′,4,4′-tetrachlorobiphenyl) enhanced the activity of 4-chlorobiphenyl hydroxylase at least 10 times more than phenobarbitone-type inducers (phenobarbitone, 2,2′,4,4′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl) enabling these two classes of inducers to be clearly distinguished. It is concluded that 4-chlorobiphenyl is preferentially metabolized by the 3-methylcholanthrene-inducible form(s) of cytochrome P-450 and that this class of microsomal enzyme inducers can be readily distinguished from phenobarbitone-type inducers by means of the 4-chlorobiphenyl hydroxylase assay.     

Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate.

This report describes the preparation of a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate and its use in a sensitive fluorometric assay of neuraminidase (EC 3.2.1.18) from Vibrio cholerae, cultured fibroblasts, and human leucocytes. V. cholerae neuraminidase showed maximum activity at pH 4.6 and an apparent K_m of 1.5 mm and was activated by CaCl₂ and inhibited by ethylenediaminetetraacetate, NaCl, and N-acetylneuraminic acid. The inhibition by N-acetylneuraminic acid was competitive (K_i = 6.1 mm). Cultured fibroblast and leucocyte neuraminidases showed maximum activity between pH 4.2 and 4.4 and apparent K_m values of 0.13 and 0.22 mm, respectively. Neuraminidase activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gels

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H₂O₂ equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe²⁺ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO₃ or SnCl₂ followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.     

A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster

Individual segments of the chromosomal DNA in D. melanogaster were isolated, and the sequences they contain were analyzed for repetition and mapped within the polytene chromosomes. Isolation was achieved by first constructing circular hybrid DNA molecules consisting of single chromosomal segments linked by poly(dA):poly(dT) joints to single molecules of the tetracycline resistance plasmid, pSC101. Tetracycline-sensitive E. coli were transformed to resistance by this heterogeneous population of hybrid molecules and homogeneous populations of different hybrids were isolated from the clones of transformants. Three hybrid plasmids (pDm 1, 2, and 4) were studied in detail. Each exhibits the structure expected from the method of construction and none exhibits internal sequence repetition detectable by reassociation kinetics. The D. melanogaster sequences in pDm2 and 4 belong to a single class defined by little or no repetition within the genome and localization to a single chromomeric region in the polytene chromosomes. The characteristics of this class, which also includes 4 of a second set of 6 hybrids, are not compatible with tandem repetition models for the chromomere. The sequences in pDm1 are repeated 90 times and are located in 15 different chromomeric regions and within the chromocentric β-heterochromatin. This distribution is of the kind predicted by certain regulatory models, for example, that of Britten and Davidson (1969).     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.     

A combined theoretical and experimental study of the interaction of metrizamide with proteins

A theory of isopycnic sedimentation of interacting systems provides a theoretical explanation for the isopycnic behavior of catalase and other proteins in metrizamide gradients of pH 7.0–7.5, particularly the bimodal banding patterns reported by some investigators. As for whether or not the model is realistic, a spectroscopic investigation provides an unambiguous demonstration of a reversible interaction between metrizamide and the catalase dimers formed by dissociation of catalase tetramer at pH 3. The metrizamide-catalase dimer complex(es) is of high bouyant density. In addition, metrizamide at pH 7.4 interacts with heat-denatured ovalbumin. These observations suggest that the denser band in the experimental bimodal isopycnic patterns might be a complex(es) of metrizamide with a comformationally altered state of the protein.