Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Distribution of Thiamine, Thiamine Phosphates, and Thiamine Metabolizing Enzymes in Neuronal and Glial Cell Enriched Fractions of Rat Brain

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.     

Transient Photoresponses of a Phototactic Microorganism, Haematococcus Pluvialis, Revealed by Light Scattering

A new method, using incoherent light scattering, has been developed to measure the flagellar beating frequency of swimming microorganisms. By means of this method, transient changes of flagellar beating frequency in response to white light flashes have been revealed in samples of a phototactic microorganism, Haematococcus pluvialis. An increase of flagellar beating frequency occurs when the flash dose (flash intensity × flash duration) is sufficient. Reciprocity between light intensity and flash duration holds for durations not exceeding 60-80 ms. For lower doses a bimodal distribution of flagellar beating frequency is revealed. No effect is observed for very low flashes or for red stimuli, whereas green light is effective. A detailed analysis of experimental results has allowed us to determine the characteristic time of the effect and follow its evolution. The correlation of this effect with visually observed behavior is discussed and a possible underlying mechanism is suggested.     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of Rhesus monkey and nephritic man

Summary Round bodies, tubular profiles and crystalline images have been studied by electron microscopy in the endothelium of seven normal young Rhesus monkeys and in the renal glomerular endothelium of two nephritic human patients. The crystalline images are most frequently formed by aggregation of round bodies, 200–240 Å in diameter. In Rhesus monkeys a variety of crystalline images are seen. On the contrary, in nephritic patients round bodies and tubular profiles are rare and less organized. In the glomerular endothelium of two normal men they were not found.The results obtained suggest that the round bodies, the tubular profiles and the crystalline images result from sectioning of a system of undulating tubules associated with the smooth endoplasmic reticulum. In nephritic patients the formation of such a tubular system could represent a change taking place within the affected cells as a pathologic response to the disease.This work was supported by U.S. Public Health Service (N.H.I.), Grant AI-04527-03-04, and by the Consiglio Nazionale delle Ricerche (C.N.R.), Contributo 115/0815/0-1365. The Authors are greatly indebted to Miss Hermina Spiele for skilful technical assistance.     

A re-evaluation of the mesangial cells of the renal glomerulus

Summary The topographical localization of the mesangial cells in renal glomeruli of rats, and their relationships with the structures at the hilus of the glomerulus were studied in this investigation. It was observed that the mesangial cells occupy a parietal position in the wall of the glomerular capillaries, and that they are anatomically continuous with the smooth muscle cells of the tunica media of the glomerular arterioles.This study was supported by a United States Public Health Service Grant AM 08628 (Institute of Arthritis and Metabolic Diseases).     

Stomatal and cuticular transpiration of beans (Phaseolus vulgaris M.) attacked by rust (Uromyces appendiculatus [Pers.] Link)

The changes of stomatal and cuticular transpiration of bean plants were investigated by graphical transpiration curves method (Slavík 1958). Bean leaves were infected by fungusUromyces appendiculatus (Pers.) Link. After the infection the intensity of stomatal transpiration had a decreasing tendency. Beginning with the sixth day after infection, the proportion of stomatal and cuticular transpirations becomes more expressive, i.e. the leaves transpire more by cuticles than by stomata. The higher share of cuticular transpiration brings extensive water relations to the diseased plants.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.