Maleate Buffer Research Articles

Release of Soluble Protein and Nitrogen in Alfalfa. II. Infleunce of Potassium and Nitrogen Fertilizer on Three Cultivars1

Reduced incidence of ruminant bloat was reported on legume pastures fertilized with high levels of potassium (K) or K plus nitrogen (N). Amount of soluble protein released from alfalfa (Medicago sativa L.) into sodium maleate buffer (pH 6.9) was proposed as possible estimate of ruminant bloat potential in comparative studies. Our objective was to determine whether K and N fertilization would influence concentration of several N fractions released from alfalfa.‘Ramsey,’ ‘Saranac,’ and ‘520’ alfalfa cultivars were fertillzed in the fall with 0 (control), 350, and 700 kg as K2SO4/ha and in the following spring with these same levels of N as NH4NO(34 3 × 3 factorial). The upper 10 to 15 cm of told‐bud stage alfalfa in first and second crops was analyzed for several N fractions.As N applied increased, mean concentrations of total N (TN), total soluble N (TSN), soluble nonprotein (SNPN), and soluble protein N (SPN) increasedm first‐harvest herbage. Mean concentrations of SPN increased 10.8% at 300 kg N/ha and 13.3% at 700 kg over that in alfalfa not fertilized with N. As the K level increased, mean concentrations of TN, TSN, and SNPN decreased in first‐harvest herbage. Fertilization with K did not affect concentration of SPN. Mean concentrations of TN, TSN, and SNPN in Saranac and TN, TSN, and SPN in 520 first‐harvest herbage were slightly but significantly greater than those in Ramsey. Cultivars did not affect concentrations of N fractions in secondharvest herbage.We found no evidence that fertilization of alfalfa with K or N reduced its apparent bloat potential, as estimated by SPN release. Instead, we showed that alfalfa fertilized with high levels of N had a slightly increased apparent bloat potential.

Effect of buffer and pH on growth and protein content of carrot (Daucus carota L.) in liquid shake culture

Phosphate, succinate, acetate and maleate buffers were tested for buffering B5 liquid medium. Phosphate (0·02 M) and acetate (0·002 M) were the most suitable buffers as they promoted dry weight accumulation and nitrogen assimilation, they also exhibited less change in pH after 5 days cultivation of carrot cells. The pH increase from 5·5 up to 6·5 also increased the dry weight accumulation and cell size; pH 7·0 was detrimental to dry weight accumulation.

New cytochemical method for adenylate cyclase and guanylate cyclase with dimethyl sulfoxide.

To assess the appropriateness of the lead nitrate method using dimethyl sulfoxide (DMSO) for the cytochemical demonstration of adenylate cyclase (ACLase) and guanylate cyclase (GCLase), we have carried out a biochemical assay of ACLase and GCLase in parallel with the cytochemical study. Tissues from rats were fixed in a mixture of 2% paraformaldehyde, 0.25% glutaraldehyde and 5% DMSO in 0.1M cacodylate buffer, pH 7.4, for 30min, and then washed with 5% DMSO in 0.1M cacodylate buffer. Vibratome or Microslicer sections were incubated in the following medium for 15-60min at 37°C. Medium for ACLase: 80mM Tris-maleate buffer, pH 7.4; 4mM MgSO4; 10mM NaF; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM AMP-PNP. Medium for GCLase: 80mM Tris maleate buffer, pH 7.4; 3mM MnCl2 or MgSO4; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM GMP-PNP. Some reaction medium for GCLase contained NaN3.ACLase and GCLase activities were biochemically and cytochemically enhanced by adding DMSO to the incubation medium as well as to fixatives. Cyclases were localized in the plasma membranes, gap junctions, endoplasmic reticulum and Golgi apparatus.

Distribution and selective extraction of filamentous components associated with axonal microtubules of crayfish nerve cord

Axons of crayfish nerve cord contain a prominent matrix network of 4- to 8-nm branched filaments which attach to and interconnect all the axoplasmic organelles, especially microtubules. The axoplasmic matrix filaments (AMF) can be selectively extracted by incubating interconnective pieces in 0.1 M Tris—maleate buffer (pH 8.6) for 1 hr at room temperature. After Tris—maleate extraction a component of the AMF remains which, when viewed in stereo, forms a spiral as it attaches to and winds around the surface of the microtubule, perhaps reflecting microtubule polarity. This microtubule-associated spiral is removed from the tubule surface by 0.4 M Pipes buffer or trypsin. We also report the cytochemical localization of ATPase activity within the axon in a distribution which parallels that of the microtubules. Based on these and other findings, a model for axoplasmic transport is presented which involves microtubules and the filamentous components of the axoplasmic matrix.

20] Nicotinamide deamidase from Flavobacterium peregrinum

This chapter discusses the formation of nicotinamide deamidase from Flavobacterium peregrinum . The first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) from nicotinamide in most microorganisms involves the deamidation of nicotinamide to form nicotinic acid and ammonia. This enzyme activity can be assayed by measuring ammonia formed colorimetrically or by measuring nicotinic acid formed or nicotinamide hydrolyzed. The standard deamidase reaction mixture contains 0.2 ml each of [7- 14 C]nicotinamide, maleate buffer, and MnCl 2 , plus deamidase to a total volume of 1.0 ml. After incubation for 15 min at 37°, the reaction is stopped by the addition of 0.5 ml of a 0.1 N acetic acid solution and heated in boiling water for 1 min. A 1.0-ml portion of the deproteinized reaction mixture is placed on a Dowex 1 X8 formate column. After the column has been washed, with 50 ml of water, the [7- 14 C]nicotinic acid is eluted, with 9 ml of 0.1 N formic acid, and the eluate is adjusted to 10 ml with water. The radioactivity recovered in this eluate is determined with a liquid scintillation spectrometer. Nicotinamide deamidase is not affected by MgCl 2 and CaCl 2 , whereas HgCl 2 , p -chloromercuribenzoate, ZnCl 2 , CuSO 4 , and FeCl 2 markedly inhibit enzyme activity. Enzyme activity is also inhibited by EDTA but not by α,α′-dipyridyl and o -phenanthroline.

Kinetics of Manganese Oxidation by Cell-Free Extracts of Bacteria Isolated from Manganese Concretions from Soil

A study of the kinetics of Mn oxidation catalyzed by cell extracts of two bacterial isolates (E(1), Pseudomonas III [new isolate] and E(4), Citrobacter freundii) isolated from the core of manganese concretions from Greek soils is presented. The reaction velocity of Mn oxidation was determined from the rate of consumption of Mn. The oxidation of Mn was followed by measuring changes in Mn concentration by activation analysis and by atomic absorption spectrophotometry. The reaction velocity was directly proportional to cell extract concentration when the reaction time was 1 h. At longer reaction times, the relationship deviated from linearity because substrate concentration became limiting. The rate of Mn oxidation increased with the Mn concentration. Analysis of the results by application of the integrated Michaelis equation for determining Michaelis constants and maximal velocities either in the presence (K(m) = 3.33 mumol/ml and V(max) = 1.25 mumol/ml.h) or in the absence of maleate buffer (K(m) = 2.52 mumol/ml and V(max) = 2.04 mumol/ml.h) indicated a strong affinity between the oxidizing system and manganese. All results in this study are consistent with an enzymatic manganese-oxidizing system and give an indication of the mechanism of biological Mn oxidation in soil which differs from that in the marine environment.

N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli.

Glycinamide ribotide transformylase from Escherichia coli was obtained free of N5,N10-methenyltetrahydrofolate cyclohydrolase activity by DEAE-cellulose chromatography. In reaction mixtures containing this enzyme preparation in potassium maleate buffer, pH 7.2, no detectable interconversion of N5,N10-methenyltetrahydrofolate occurred. Upon addition of glycinamide ribotide, N-formylglycinamide ribotide was formed when N10-formyltetrahydrofolate was present; no formylation occurred in the presence of N5,N10-methenyltetrahydrofolate. A method for the synthesis and purification of glycinamide ribotide is presented.

A Possible Role for Leaf Cell Rupture in Legume Pasture Bloat1

Leaves from seven forage crop species were crushed in a mortar and pestle to demonstrate a relationship between susceptibility to mesophyll cell rupture and the tendency to cause bloat in ruminant animals. The bloat‐causing forages were alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), and white clover (T. repens L.). The bloat‐safe species were cicer milkvetch (Astragalus cicer L.), birdsfoot trefoil (Lotus corniculatus L.), sainfoin (Onobrychis viciaefolia Scop.), and bromegrass (Bromus inermis Leyss.). Two grams of fresh leaves were crushed between two layers of 100‐mesh nylon cloth in a mortar and pestle. The leaf material which passed through the nylon cloth was suspended in 0.2 M sodium maleate buffer (pH 6.9) and allowed to separate into sediment and supernatant fractions by standing at 4 C for 45 min. The sediment fraction contained intact mesophyll cells and the supernatant fraction contained fragments of chloroplast lamellae which had been released from ruptured mesophyll cells. Compared to the bloat‐causing legumes, the bloat‐safe legumes had 10 to 20 times more whole mesophyll cells released from the leaf tissue. Conversely, the supernatant fractions from the bloat‐causing legumes contained 6 to l0 times more chlorophyll than supernatant fractions from the bloat‐safe legumes. These data showed that mesophyll cells in the bloat‐safe forage legumes were more resistant to mechanical rupture compared to mesophyll cells in the bloat‐causing forage legumes. Disruption of sainfoin leaves in buffer containing 10% polyvinylpyrolidone did not affect the number of whole mesophyll cells in the filtrate fraction. Hence the presence of tannins and leaf cell resistance to rupture are two separate and complementary explanations for the bloat‐safe nature of sainfoin. The bloat‐safe nature of cicer milkvetch, which does not contain tannins, was attributed to the resistance to rupture of the mesophyll cell walls.

Purification and properties of a triacylglycerol lipase from Mycobacterium phlei

In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified 800-fold from stationary phase cells of Mycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, pI. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. With phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent K m of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested ( C 2– C 18), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p-nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.

Experiments with beta-chain variants of the hemoglobin of Mus musculus.

Starch gel electrophoretic and ultracentrifuge methods failed to demonstrate any differences between the hemoglobins of mice of the Shanghai and HBBP/Cag strains and crosses among these strains. The apparent identity of these hemoglobins is thought to stem from the contribution of Asian mice to the British mouse fancy from which the laboratory strains have Hbb-p in part descend. Maleate buffer of pH 7 or above can be used to prevent the formation of disulfide-bridged dimers of mouse hemoblobins. However, the minor electrophoretic bands of Hbb-p and Hbb-d react with approximately twice as much maleate as the major bands of each of these hemoglobins, although the minor bands like the major contain only one free cysteine group per beta chain. This can be explained by the alkylation of the epsilon-amino of lysine residue beta76, but some evidence for the alkylation of histidine in the minor band of Hbb-p is also presented.

Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions.

The enzyme, CTP:phosphatidate cytidylyltransferase (EC2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer-detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleolphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60mM for MG2+, 6MM for CTP and 250 mug per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90,000 X g fraction (microsomal) with small but significant activity in the 12,000 X g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses); 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl-(3.85), 1-stearoyl-2-arachidonoyl-(3.31), 1-arachidonoyl-2-stearoyl-(3.16), 1,2-diarachidonoyl-(0.72), 1,2-dicaproyl-(0.67), 1,2-dipalmitoyl-(0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.

The temperature-sensitive adenylylation of tRNA in cacodylate and other buffers

A temperature-sensitive adenylylation of tRNA (in the presence of cacodylate buffer) by an enzyme from Neurospora crassa has been documented. Notably different extents of adenylylation of tRNA are achieved at 20 and 37°C in the presence of excess enzyme. The phenomenon is not due simply to “renaturation” of metastable forms of tRNA in the presence of Mg 2+ at the warmer temperature. The phenomenon is apparent with both homologous and heterologous tRNAs. This effect is not shown in Tris—maleate and certain other buffers. Cacodylate ion, like arsenate and phosphate, appears to play some role other than mere binding of Mg 2+ in a form inaccessible to the enzyme; rather, a Mg 2+ — cacodylate chelate may exert an effect itself. Possibly, a “mixed chelate, formed between one cacodylate ion and Mg 2+ with the phosphate group of the tRNA, may so alter the structure of tRNA that it is no longer accessible to this enzyme. The inactive complex, whatever its composition, is stable at 20°C but breaks down rapidly at 37°C.

Carboxytransphosphorylase: VIII. LIGAND-MEDIATED INTERACTION OF SUBUNITS AS A POSSIBLE CONTROL MECHANISM IN PROPIONIBACTERIA

Abstract Phosphoenolpyruvate carboxytransphosphorylase from Propionibacterium shermanii catalyzes the conversion of P-enolpyruvate, orthophosphate, and CO2 to oxalacetate and pyrophosphate. The enzyme occurs in three enzymatically active forms, tetramer, dimer, and monomer, but only the tetrameric and monomeric forms have been isolated from the bacteria. The dimeric form was initially observed by Haberland et al. (Haberland, M. E., Willard, J. M., and Wood, H. G. (1972) Biochemistry 11, 712–722) in reacting enzyme sedimentation centrifugation, and it was shown that dissociation of the tetrameric enzyme and association of monomeric enzyme to dimeric forms occurred during catalysis of the forward reaction. We have now shown that the association-dissociation is not caused by the substrates but by the product, oxalacetate. When the forward reaction is conducted in the presence of hydrogen peroxide, which oxidizes oxalacetate to malonate, no dimer formation is observed. In addition to oxalacetate, malate and fumarate induce the subunit interactions. Our results show that the dimeric species is less active than the tetrameric species. Preincubation of the tetrameric enzyme at dilute concentrations in malate buffer results in a loss of 50% of the activity within 5 min. This loss in activity is strongly dependent on the concentration of protein, there being less loss of activity with higher concentrations of protein in the malate buffer. The tetramer-dimer interaction is in equilibrium since only a single peak is observed on centrifugation in malate buffer, and as the protein concentration of the tetrameric form is decreased the s20, obs value decreases until it approaches that of the dimer. At the same time, there is a loss in enzymatic activity which correlates with the increase in the dimeric form. The monomeric species has a specific activity about one-fourth that of the tetrameric species, and the activity is not affected by the presence of malate although there is total conversion of monomeric into dimeric species. The continued presence of an effector is required for maintenance of the dimeric species from either tetramer or monomer. Sedimentation equilibrium studies in malate buffer gave a molecular weight of 224,000 for the dimeric species formed from monomeric enzyme. When tetrameric enzyme was employed a nonlinear log Y versus r2 plot was observed reflecting the equilibrium between tetramers and dimers induced by the presence of an effector. The malate-treated tetrameric enzyme is more susceptible to proteolytic attack by trypsin and chymotrypsin than the untreated enzyme. It is proposed that the ligand-induced subunit interactions play a role in the control of the propionic acid fermentation.

Renin activity by radioimmunoassay: The plasma incubation step

1. We have investigated several plasma incubation protocols by which angiotensin I can be generated for radioimmunoassay, including those employed by 3 leading commercial renin activity kits. 2. Without buffering, (kits No. 1 and 2), the pH of human and rat plasma rises to 8, or above, within 1 hour of incubation at 37°C, even if initially adjusted to 6 as recommended by some workers. Commonly, angiotensin generation rate declines within 1–2 hours, invalidating standard 3 hour incubations. 3. Plasma diluted with as little as 1 volume of various buffers generates significantly less angiotensin than more concentrated plasma (0.1 volume dilution). Dilution with 2 volumes of maleate buffer, pH 6, final molarity 0.133 (kit No. 3) controls pH satisfactorily, but yields suboptimal amounts of angiotensin. 4. Phosphate buffer, 0.1 or 1.0 volume, 0.05–0.15 M in the incubate, pH 6, provides pH control and generally higher angiotensin generation than in the presence of comparable molarities and dilutions of phosphate or Tris buffers adjusted to pH 7.4. 5. Successful buffering does not guarantee linear angiotensin production within a predetermined incubation period. We propose the use of sequential incubation intervals, as short as 15 minutes, in order to establish a region of linearity from which more accurate estimates of plasma renin activity can be made in humans and rats.

EFFECTS OF BICARBONATE REMOVAL ON THE SPONTANEOUS MECHANICAL ACTIVITY OF THE GUINEA PIG UTERUS

1. Effects of bicarbonate removal and of decreasing external pH on the mechanical and electrical activities in isolated strips of the guinea pig uterus were investigated.2. When bicarbonate was reduced at 37°C, a high degree of contractile activity was observed, which was associated with high frequency spike activity. However, when the same experiment was repeated at 21°C, the activity was suppressed. These sustained contractions were produced whether the HCO-3-free solution was acid (pH: about 6) in the presence of 3% CO2, or the solution was alkaline (pH: about 8) in the absence of CO2.3. When the pH of the solution was decreased to 6 by 5mM tris malate buffer, the mechanical activity was also increased in the presence of 15.5mM HCO-3.4. In low Cl- (7 mm) Krebs solution the mechanical activity depended on the substitute for Cl-: substitution with isethionate reduced or stopped the activity, while substitution with NO-3 increased the activity. The increase in activity on HCO-3 removal was less in isethionate solution and much larger in NO-3 solution.5. At a lower temperature (21°C), HCO-3 removal suppressed the activity when O2 containing 3% CO2 was bubbled through the solution.6. It is suggested that HCO-3 removal and acidification increase Cl-permeability of the membrane and that this leads to high spontaneous activity. It is also possible that modification of the external buffer system may affect the internal pH and metabolism, which in turn modifies spontaneous activity.

Studies on partially purified pig liver steroid delta 4-5 beta-reductase activity.

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

Purification and Properties of Nicotinamide Deamidase from Flavobacterium peregrinum

Abstract Nicotinamide deamidase from Flavobacterium peregrinum was purified about 210-fold with the use of protamine and MnCl2 treatments, acetone fractionation, ammonium sulfate fractionation, Sephadex G-200, and Cm-Sephadex column chromatography. The optimum pH of the purified nicotinamide deamidase is 6.5, and the Km value for nicotinamide is 0.2 µm. The molecular weight was estimated by means of gel filtration to be 48,000. The enzyme activity of the crude preparation was increased by 2.5-fold by addition of Mn2+ at a concentration of 10 mm followed by incubation at 37° for 30 min. The purified enzyme preparation was inactivated by dialysis in the absence of Mn2+ and no recovery of enzyme activity was possible even when Mn2+ or Mn2+ plus cysteine was added to the reaction mixture. When the enzyme was dialyzed overnight in the presence of Mn2+, almost all of the enzyme activity was retained. A dialysis of the enzyme against 0.01 m maleate buffer (pH 6.5) containing 5 µm HgCl2 resulted in complete retention of enzyme activity when 50 mm cysteine was added to the reaction mixture. CuSO4 and SnCl2 also showed effects similar to that of HgCl2, but their potency for enzyme stability was small. Mg2+, Ni2+, Zn2+, and Fe2+ did not show any effect on enzyme activity. All the nicotinamide analogues having a trivalent nitrogen in the pyridine ring showed a competitive inhibition of nicotinamide deamidation, whereas compounds having a tetravalent nitrogen seemed to have no effect. From these results the structure of the enzyme-metal-substrate complex was proposed as [see PDF for equation]

Partial Resolution of the Enzymes Catalyzing Photophosphorylation: XI. MAGNESIUM-ADENOSINE TRIPHOSPHATASE PROPERTIES OF HEAT-ACTIVATED COUPLING FACTOR I FROM CHLOROPLASTS

Abstract Conditions are reported under which purified coupling factor 1 from spinach chloroplasts, suitably activated, exhibited Mg++-dependent ATPase activity at rates of about 15 µmoles of Pi released per mg of protein per min. The presence of a carboxylic acid and proper MgCl2 concentrations were the main factors determining the rate of hydrolysis. At pH 6, with 8 mm MgCl2, sodium maleate accelerated the Mg++ATPase activity of coupling factor 1 up to 30-fold. At pH 8, 2 mm MgCl2 was optimal and bicarbonate was more effective than maleate as an activator. Higher concentrations of MgCl2 inhibited the reaction. A monospecific antibody against coupling factor 1 inhibited both the Mg++- and the Ca++-dependent ATPase activities. The specificity for triphosphonucleotides was similar for the two reactions. The Km of the Mg++ATPase for ATP was 0.1 mm at pH 6 with 2-(N-morpholino)ethane sulfonic acid buffer and 1.1 mm with maleate buffer. It is concluded that carboxylic acids increase the Vmax by removing inhibitory products of the reaction. From the mode of inhibition by ADP, existence of at least two active sites acting cooperatively, is proposed.

A new assay of phosphorylase based on the filter paper technique

A rapid method for the assay of phosphorylase has been developed, similar in technique to the method described by Thomas et al. (8) for glycogen synthetase. It utilizes the ability of phosphorylase to incorporate 14C-labeled glucose from 14C-glucose 1-phosphate into glycogen, which is then precipitated on filter paper squares by immersion in ice cold 66% ethanol. After removal of surplus 14C-glucose 1-phosphate, the papers are counted directly in a vial containing scintillation fluid. Several buffers commonly used in phosphorylase assays were compared and it was found that measuring in a 100 m M maleate buffer gave approximately 15% lower activity than assaying in any of the other seven buffer systems tested.

Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

A method for ATP-ADP determination in plasma by ethanol extraction and firefly luminescence has been modified for platelets. EDTA had to be included in the ethanol for proper inactivation of platelet adenylate kinase. Presence of EDTA inhibited conversion of ADP to ATP (prior to luminescence measurement), presumably through a drop in pH caused by the interaction between H 2EDTA 2− and the Mg 2+ of the pyruvate-kinase system employed. Introduction of Tris maleate buffer gave 100% ADP-ATP conversion. Optimal amounts of platelet ATP (99%) and ADP (80–90%) were solubilized by 50% EDTA ethanol; less EDTA ethanol failed to inactivate ATP-ADP degrading platelet enzymes whereas increasing EDTA ethanol concentrations gave decreasing solubilization of the nucleotides. ATP and ADP could be extracted from the EDTA ethanol-insoluble material by 0.6 M HClO 4. ADP and ATP added to such EDTA-ethanol and HClO 4 extracts of both platelet-rich plasma and suspensions of washed platelets gave luminescence responses per mole nucleotide equaling that of ATP-ADP standards. Optimal conditions for nucleotide determination are described as well as the beneficial use of the DuPont luminescence biometer.