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Biomedical subjects

L C Davis

Publications and source records attributed to L C Davis.

At least 37 records · Page 2Linked to original sources
PubMed1984

Chemically coupled spectrophotometric assays based on flow injection analysis: determination of nitrogenase by assays for creatine, ammonia, hydrazine, phosphate, and dithionite.

Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of 1-25 microliters are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 microliters/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomoles of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 ml per hour; typical sample throughout is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenase, continuously monitoring creatine produced from creatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehyde-quenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenase reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde.

Ammonia↗
PubMed1984

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules.

I have measured acetylene diffusion through plant tissues including nodules from several species of legume-vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O(2) where there is no N(2) reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.

Journal Article↗
PubMed1983

Training community-based, Asian-American mental health personnel in behavior modification.

Sixteen Asian-American and Pacific Island mental health professionals received 10 weekly, 2-hour training sessions on behavior modification and its application, in particular, to Asian-American clients. A three-fold assessment of the training results revealed: a) a significant increase in knowledge of behavior modification principles, b) a significant increase in ability to accomplish a behavioral analysis and behavioral treatment plan, and c) a significant increase in one of three attitudes toward behavior modification: oriented activity. In general, the training was rated highly and, perhaps more importantly, was viewed as having relevance for this particular minority population which often under utilizes mental health services. The training model and needs for further consultation and supervision were discussed.

Asian↗
PubMed1983

Interaction of nitrogenase with nucleotide analogs of ATP and ADP and the effect of metal ions on ADP inhibition.

The interaction of a large number of ATP and ADP analogs with nitrogenase from Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianum has been examined. Only 1,N6-etheno-ATP and 2'-deoxy-ATP served as substrates for acetylene reduction. Other triphosphates including GTP, ITP, 8-Br-ATP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, 6-chloropurine riboside triphosphate, and AMP-PNP were inert, showing less than 50% inhibition at levels up to two- to fivefold greater than ATP. Xanthosine triphosphate behaved simply as a chelator of magnesium, activating the enzyme at low levels but strongly inhibiting at high levels. When nucleotide diphosphates were tested as inhibitors with enzyme from A. vinelandii, GDP, dGDP, and 6-chloropurine riboside diphosphate were ineffective, XDP was three- to fivefold less effective, and dADP and 1,N6-etheno-ADP were about equally as effective as ADP. With enzyme from C. pasteurianum, dADP was twofold less effective than ADP, XDP was fivefold less effective, and IDP and 1,N6-etheno-ADP appeared to be ineffective. Results with enzyme from K. pneumoniae were very similar to those obtained with A. vinelandii. Different metal ions were tested in the presence of both ATP and ADP to determine whether preferential binding to one nucleotide or the other might alter the ADP/ATP ratio needed for 50% inhibition of activity. Magnesium and manganese gave the same ratio, while with Fe and Co, slightly less ADP was required for equivalent inhibition. Nickel appeared to reduce the sensitivity of A. vinelandii nitrogenase to ADP inhibition while increasing that of C. pasteurianum, but both effects were less than twofold. Calcium, strontium, and aluminum ions were inert with enzymes from these organisms. Cd and Zn were also ineffective with K. pneumoniae. Two isomers of ATP beta S were prepared by enzymatic synthesis from ADP beta S. The A form was a more potent inhibitor of A. vinelandii nitrogenase.

Adenosine Diphosphate↗
PubMed1983

Training Mexican American mental health personnel in behavior therapy.

Fourteen Mexican American mental health workers were trained in basic behavior therapy principles and behavior contracting in 10 weekly, 2 hr sessions. A significant increase was found in knowledge of principles, and attitudes toward behavior therapy became more positive. In addition, clinical skill in doing a behavior analysis and formulating a behavior therapy plan also improved. A 6 month follow-up suggested continued application of training. Results and match of behavior therapy training to differing cultures are discussed.

Behavior Therapy↗
PubMed1983

Broad-zone active-enzyme chromatography. Keto-acid dehydrogenases as associating systems.

We have extended the method of active-enzyme chromatography to include the use of broad zones of enzyme. This allows examination of interacting systems in a way formally analogous to sedimentation velocity so that simulation of the observed activity profiles is possible. The method has been applied using pyridine nucleotide-linked active enzyme assays. At the concentrations presently accessible by this technique, hexokinase and glucose-6-phosphate dehydrogenase, both associating systems, show single symmetrical boundaries, as does isolated diaphorase, while pyruvate and alpha-ketoglutarate dehydrogenases show more complex patterns, with the position of the reaction boundary for diaphorase activity being dependent on enzyme concentration.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Sugar and organic Acid constituents in white clover.

Major ethanol-soluble carbohydrate and organic acid constituents of white clover (Trifolium repens) have been identified by use of high-performance liquid chromatography and gas chromatography. In leaves, petioles, roots, and nodules, pinitol (3-O-methyl chiro-inositol) is the predominant sugar, with sucrose present in lower concentration. In leaves and petioles there are significant levels of alpha- and beta-methyl glucosides, linamarin, glucose, and fructose. In the nodules glucose is rarely present at detectable levels. The concentration of pinitol is generally greater than 25 millimolar in each tissue examined whereas the level of sucrose varies depending on the time of day. Sucrose is the major sugar significantly labeled during 1 hour administration of (14)CO(2) and accounts for more than 99% of all the radioactivity detected in the nodules at early times. Between 3 and 7 hours after labeling, 6% of the radioactivity is found in the organic acids fraction and 5% in the basic fraction of nodules. Malonic acid does not appear to be present in unusually high concentrations in either leaves or nodules of white clover.

Journal Article↗
PubMed1980

Estimating Kinetic Parameters when the Amount of Enzyme Added to an Assay Is Not a Controlled Variable: NITROGENASE ACTIVITY OF INTACT LEGUMES .

Reliable estimates of Michaelis constants (K(m)) and inhibitor constants may be obtained, in the absence of control over the amount of enzyme being added to any assay system, provided the following constraints are met. Michaelis-Menten kinetics are obeyed. Two rate measurements must be made with the same sample of enzyme: at low and high substrate concentration for determining K(m) or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation [Formula: see text] Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the K(m) has been calculated. The above technique has been applied to study of the acetylene-reducing ability of intact legume plants. The apparent K(m) for acetylene reduction by nitrogenase in legume nodules is approximately 1/100 atmosphere in the absence of nitrogen and approximately 1/40 atmosphere in its presence.

Journal Article↗
PubMed1980

In vivo and in vitro kinetics of nitrogenase.

We measured some of the kinetic parameters of nitrogenase to intact systems of Clostridium pasteurianum and Klebsiella pneumoniae to compare them with the kinetics of the enzyme in vitro. We found that the enzyme showed multiple apparent Km values for acetylene reduction in vivo, as it does in vitro. Carbon monoxide was a noncompetitive inhibitor of acetylene reduction; azide was a noncompetitive inhibitor of acetylene reduction, and nitrogen was a partial inhibitor of acetylene reduction. Cyanide was a noncompetitive inhibitor of acetylene reduction in C. pasteurianum but it was a metabolic poison in K. pneumoniae, in addition to being an inhibitor of nitrogenase. The partial nature of nitrogen inhibition was apparent in assays where both nitrogen and CO were present. Nitrogen did not alter the apparent Ki for CO, nor did the presence of CO enhance the competitive effectiveness of nitrogen. By using recombined nitrogenase fractions, we found that the ability of nitrogen to inhibit hydrogen evolution or acetylene reduction varied with the ratio of protein components. The in vivo inhibition of acetylene reduction by dinitrogen was comparable to that obtained with an excess of the Fe protein in vitro. We conclude that there is an effective excess of the Fe protein available under active growth conditions in vivo.

Acetylene↗
PubMed1979

Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state.

Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98, and 1.92 with t1/2 approximately 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1, and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0, and 1.98 is observed. The use of Azotobacter nitogenase components enriched selectively with 57Fe or 95Mo, as well as the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of high-potential iron-sulfur proteins and ferredoxins, respectively. The "no-CO" signal may be either an additional center or the N2-reducing site with H2 bound competitively.

Azotobacter↗