Comparison of lactate and malate dehydrogenases: Fluorescence and thermodynamic properties

Equilibrium, thermochemical, and time-resolved fluorescence measurements have been carried out in order to compare pig heart lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (MDH). The differences in the thermodynamic parameters for binding of NADH and NAD+ show the same pattern for both enzymes. The stronger binding of NADH is entropy-based, which can be understood as reflecting electrostatic interactions. The tryptophan fluorescence of MDH and LDH differ for the free enzymes and in quenching by NADH. The differences can be accounted for in terms of a single long-lived tryptophan residue present in LDH and not in MDH.     

Isolation and characterization of tox mutants of corynebacteriophage beta.

Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct form the structural gene for toxin, and that the CRM- strains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed.     

Studies on nitrosamine metabolism I. Subcellular distribution of radioactivity in tumor-susceptible tissues of RFM mice following administration of [14C] dimethylnitrosamine

The distribution of radioactivity in tumor-susceptible (liver and lung) and non-tumor-susceptible (heart, forestomach, and esophagus) tissues of male RFM mice was investigated at timed intervals following a single intragastric administration of ¹⁴C-labeled DMN.³ The greatest amount of radioactivity was associated with the tumor-susceptible tissues—liver and lung. At 15 min, the relative amount of radioactivity in the homogenates of heart, forestomach, esophagus, livers and lung was 1, 2, 3, 10, and 70, respectively. The AS components of lung contained about six times as much radioactivity as the liver 15 min after administration; at 16 hr, the level of radioactivity had decreased and was equal in amount. The AI components of both tumor-susceptible tissues incorporated much less radioactivity than the AS components, indicating that only a small amount of methyl label is covalently bound to cellular macromolecules. The amount or radioactivity in the AI components ranged from 2–34% in the lung and from 11–33% in the liver. In the lung C-fraction the range of radioactivity was 75–89% for the AS components and 52–74% for the AI components. The radioactivity in the AS components of liver C-fraction ranged from 50–89%, and from 52–68% for the AI components. The results suggest differences in the affinity, transport, and/or metabolism of DMN between liver and lung, as well as between tumor-susceptible and non-tumor-susceptible tissues.     

THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.     

Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide.

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Mg induced Ca deficiency under alkaline conditions

Little is known about Mg induced Ca deficiency in alkaline conditions, and the relationship between Mg induced Ca deficiency and Na induced Ca deficiency. Dilute nutrient solutions (dominated by Mg) were used to investigate the effect of Ca activity ratio (CAR) on the growth of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald). At pH 9.0, root growth was reduced below a critical CAR of 0.050 (corresponding to 90% relative root length). Root growth was found to be limited more in Mg solutions than had been previously observed for Na solutions. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for both Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present.     

Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.