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.     

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.     

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).     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone

Experiments were performed to determine if elevation of cumulus cell cAMP results in an increase in mouse oocyte cAMP while the heterologous gap junctions were intact. Both follicle-stimulating hormone (FSH) and cholera toxin induced a marked increase (>20-fold) in intracellular cAMP in isolated mouse cumulus cell-oocyte complexes in the presence of 3-isobutyl-1-methyl xanthine (IBMX). Concomitantly, both FSH and cholera toxin transiently inhibited resumption of meiosis of cumulus cell-enclosed but not denuded oocytes. The transient nature of the inhibitory effect produced by either FSH or cholera toxin was correlated with the cAMP level in the cumulus cell-oocyte complex. The inhibitory effect, however, was apparently not due to movement of cumulus cell cAMP to the oocyte via the functional heterologous gap junctions between cumulus cells and the oocyte. Radioimmunoassay of cAMP in oocytes free of attached cumulus cells or cumulus cell-enclosed oocytes exposed to either FSH or cholera toxin revealed that both groups of oocytes contained similar amounts of cAMP (about 0.14 fmole/oocyte). Metabolic labeling of cumulus cell-oocyte complexes with [³H]adenosine followed by incubation with either FSH or cholera toxin resulted in a marked increase in the amount of radiolabeled cAMP compared to that in unstimulated complexes. However, similar amounts of radiolabeled cAMP were found in oocytes derived from either stimulated or unstimulated complexes. Thus, we have not detected, using two methods of assay, that increasing the cAMP content of the cumulus cells results in any increase in the cAMP content of the oocyte. The apparent compartmentalization of cumulus cell cAMP elevated in response to either FSH or cholera toxin was not due to disruption of intercellular communication between the two cell types during the incubation; metabolic cooperativity was present between the two cell types and molecules of similar molecular weight and charge relative to that of cAMP were rapidly equilibrated between the two cell types. Testosterone potentiated the FSH/cholera toxin-induced transient inhibition of maturation of cumulus cell-enclosed oocytes. However, testosterone did not increase cAMP accumulation produced by either FSH or cholera toxin, decrease the rate of cAMP degradation, or promote movement of cumulus cell cAMP to the oocyte. Since cAMP elevated in response to FSH or cholera toxin appeared to be compartmentalized to cumulus cells and since neither FSH, cholera toxin, nor testosterone inhibited resumption of meiosis in denuded oocytes, it appears that the inhibitory effect promoted by FSH or cholera toxin is directly mediated by an agent other than cAMP, although cAMP generation is required for its action and that cumulus cells mediate the inhibition. These results are discussed in terms of a possible role of cAMP and steroids in regulating maturation in the mouse.     

Polyadenylation of nascent RNA during the embryogenesis of Ilyanassa obsoleta.

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated $\text{32}\text{64}$ cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

The role of calcium in the induction of ornithine decarboxylase in rat HTC cells

The possible role of calcium ions in the induction of ornithine decarboxylase (ODC) in rat hepatoma cells in culture (HTC) has been investigated by manipulating cellular calcium levels as follows: a) use of the calcium chelating agent EGTA to inhibit induction of ODC by dibutyryl cyclic AMP (cAMP), b) addition of Ca⁺⁺ to reverse the inhibition of cAMP induction of ODC by EGTA, c) use of a calcium ionophore in the presence of Ca⁺⁺ to induce ODC. In each case there was positive evidence for the participation of Ca⁺⁺ in the induction of ODC.     

A simple, automated apparatus for the rapid multiple flushing of reaction (assay) vessels with gases

Folin and Ciocalteu's phenol reagent may be reduced by a variety of compounds, which therefore interfere with the Lowry method of protein determination (1,2). Peters and Fouts (3) reported that two of the zwitterionic biological buffers described by Good et al. (4) reacted strongly with the Folin reagent and thus seriously interfered with protein determinations. The zwitterionic buffers have many properties which lead to their use in biological work, where protein estimations will be required. Since the publication of Good et al. (4), the range of zwitterionic buffers has been increased. The possibility that these other buffers may also produce artefacts has therefore been investigated.     

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals

The effects of hemicholinium-3 (HC-3) or 4-(1-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 μg HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP resulted in a further reduction in ACh. Pretreatment with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR) against soman was increased from 0.8 to 7.3. These data are consistent with the hypothesis that excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.     

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.     

Allethrin interactions with the nicotinic acetylcholine receptor channel

Interactions of the synthetic pyrethroid allethrin with the nicotinic acetylcholine (ACh) receptor/channel were studied in membranes from Torpedo electric organ. Allethrin did not inhibit binding of [3H]ACh to the receptor sites, but inhibited noncompetitively binding of [3H]perhydrohistrionicotoxin ([3H]H12-HTX) to the ionic channel sites in a dose-dependent manner. The inhibition constant (Ki) of [3H]H12-HTX binding in absence of receptor agonist was 30 micro M, while in presence of 100 micro M carbamylcholine it was 4 micro M. This inhibitory effect of allethrin had a negative temperature coefficient. The high affinity binding of allethrin to the channel sites of the nicotinic ACh-receptor may be indicative of a postsynaptic site of action for pyrethroids, in addition to their known action on the sodium channel.     

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [³H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol(trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).     

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.     

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.     

Allosteric interactions of the membrane-bound acetylcholine reception: kinetic studies with alpha-bungarotoxin.

The specific and irreversible reaction of a snake neurotoxin, α-bungarotoxin, with the acetylcholine receptor of electroplax membrane preparations from $\text{Electrophorus electricus}$ proceeds by an initial fast phase followed by a slower one. The fraction of the reaction in the fast phase increases with increasing initial toxin concentrations, while the fraction going slowly decreases correspondingly. Both phases are affected by compounds which initiate or inhibit nerve impulse transmissions. The time course of the reaction can be fitted to the sum of two exponentials. The dependence on initial toxin concentration of the two exponentials, and of the fraction of reaction governed by the exponentials, can be fitted to a minimum reaction mechanism which involves at least two types of toxin binding sites with different dissociation constants and ligand-induced conversion of one type of site into the other. The mechanism is consistent with our previous data which showed that activators and inhibitors of membrane electrical potential changes occupy separate sites, only half of which interact. This type of mechanism has been seen in a number of allosteric regulatory enzymes.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.     

Rabbit-liver phosphorylase: improvement of the purification procedure and assay of the inactive b form

Continuous electrophoresis buffers are described for polyacrylamide gels at pH values ranging from 3.8 to 10.2. The buffers consist of an acidic and a basic component with pK values near the pH of the buffer. The pH is maintained to within 0.5 pH unit in the electrode compartments during prolonged electrophoresis. Some proteins produce clear bands on gels with each of the 10 buffers. The buffers provide an expansion of the pH range of gel electrophoresis and are likely to be useful in the detection of genetic variation in proteins and in other applications.     

A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles

A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%.