Irwin P. Ting

Preparation of cellular plant organelles from spinach leaves

Abstract Broken chloroplasts, intact chloroplasts, mitochondria, and microbodies were isolated and purified from spinach leaf tissue using a sequential sedimentation rate-equilibrium density (S-ϱ) centrifugation technique. Organelle pellets (1000g and 3000g) were prepared in 0.5 m sucrose, 1 m m EDTA, 1 m m 2-mercaptoethanol, and 0.1% bovine serum albumin all in 0.05 m Tris buffer, pH 7.5. The organelle pellets were separately layered on linear sucrose-density gradients of 40–80% ( w v ) and centrifuged for 3 hr at 25,000 rpm (ω2t = 74 × 109 rad2 · sec−1) in an ultracentrifuge. The 1000g pellet resolved into broken chloroplasts (without outer envelope and stroma), intact chloroplasts (with outer envelope and stroma) plus mitochondria, and microbodies; the 3000g pellet resolved into broken chloroplasts, mitochondria, and microbodies. Electron micrographs and marker enzymes revealed a high degree of purity. Markers used were glycolate oxidase and slow particulate malic dehydrogenase for microbodies, cytochrome c oxidase and fast particulate malic dehydrogenase for mitochondria, NADP glyceraldehyde 3-phosphate dehydrogenase and chlorophyll for intact chloroplasts, and chlorophyll for broken chloroplasts. Estimates of purity using the enzyme markers suggested that the intact chloroplast fraction was about 13% contaminated by mitochondria and 3.5% by microbodies. The mitochondrial fraction was contaminated about 12% with microbodies and was essentially free from chlorophyll. The 3000g microbody fraction was essentially pure. The estimated isopycnic densities for the organelles were: broken chloroplasts, 1.17; intact chloroplasts, 1.21; mitochondria, 1.21; and microbodies, 1.25 g/cm3. When estimated by centrifugation in a 25–50%. ( w v ) sucrose gradient, the sedimentation-rate orders were: intact chloroplasts > broken chloroplasts > microbodies > mitochondria.

Separation and detection of organic acids on silica gel

Abstract Thin-layer chromatography of water soluble organic acids on silica gel H with a water-saturated diethyl ether-formic acid (7:1) developing system is a rapid, satisfactory qualitative method of analysis. In combination with good extraction procedures, the technique should prove particularly useful for the separation of specific groups of acids and for the detection of specific metabolic products or reactions. Particularly advantageous is the quantitative determination of radioactively labeled organic acids by gel removal and liquid scintillation counting. The formation of molybdenum blue by reduction of silicomolybdic acid on silica gel is a sensitive indicator for some organic acids and certain other organic compounds.

Malate dehydrogenases of leaf tissue from Spinacia oleracea: Properties of three isoenzymes

Abstract Starch-gel electrophoresis of leaf homogenates from Spinacia oleracea indicates four forms of malate dehydrogenase ( l -malate: NAD oxidoreductase, EC 1.1.1.37). Three of the isoenzymes (viz., microbody malate dehydrogenase, mitochondrial malate dehydrogenase, and soluble1 malate dehydrogenase) were completely separated by DEAE-cellulose anion-exchange column chromatography while a fourth isoenzyme (viz., soluble2 malate dehydrogenase) still contained soluble1 malate dehydrogenase as a contaminant. The electrophoretic and chromatographic properties of the isoenzymes are significantly different. Of the three resolvable isoenzymes, soluble1 malate dehydrogenase and microbody malate dehydrogenase are similar and differ from mitochondrial malate dehydrogenase in terms of pH-dependent substrate inhibition by oxaloacetate and in terms of their pH-dependent Michaelis constants (oxaloacetate). Soluble1 malate dehydrogenase and microbody malate dehydrogenase are inhibited less by oxaloacetate at pH 9.0 when compared to pH 6.0 or 7.5, while mitochondrial malate dehydrogenase is rather constant in its susceptibility to oxaloacetate inhibition at the same pH values. The oxaloacetate Michaelis constants of all the isoenzymes increase with increasing pH, however, soluble1 malate dehydrogenase and microbody malate dehydrogenase show a sharp increase at pH 9.0 which is not nearly so pronounced with mitochondrial malate dehydrogenase. The thermal stabilities of the isoenzymes are significantly different: soluble1 > mitochondrial > microbody. Based on gel filtration, the molecular weights of all four isoenzymes were estimated to be approximately identical, at 66,000. NAD analog experiments also suggested significant differences among the isoenzymes.

Phosotynthesis in hemiepiphytic species of Clusia and Ficus

SummaryHemiepiphytic species in the genera Clusia and Ficus were investigated to study their mode of photosynthetic metabolism when growing under natural conditions. Despite growing sympatrically in many areas and having the same growth habit, some Clusia species show Crassulacean acid metabolism (CAM) whereas all species of Ficus investigated are C3. This conclusion is based on diurnal CO2 fixation patterns, diurnal stomatal conductances, diurnal titratable acidity fluctuations, and δ13C isotope ratios. Clusia minor, growing in the savannas adjacent to Barinas, Venezuela, shows all aspects of Crassulacean acid metabolism (CAM) on the basis of nocturnal gas exchange, stomatal conductance, total titratable acidity, and carbon isotope composition when measured during the dry season (February 1986). During the wet season (June 1986), the plants shifted to C3-type gas exchange with all CO2 uptake occurring during the daylight hours. The carbon isotope composition of new growth was-28 to-29‰ typical of C3 plants.

Malic dehydrogenases in corn root tips.

Abstract Three malic dehydrogenase isozymes isolated from 2.5-day-old corn root tips were separated by anion-exchange column chromatography. By pH-dependent properties and electrophoretic mobility, the two dominant isozymes were shown to correspond to a soluble-malic dehydrogenase and a mitochondrial-malic dehydrogenase. The soluble form differed from the mitochondrial form by showing greater oxaloacetate substrate inhibition at low pH and less at high pH. The soluble enzyme was more active than the mitochondrial enzyme with the nucleotide analog, thionicotinamideadenine dinucleotide. Oxaloacetate Michaelis constants for both isozymes increased with pH with a definite change in slope at about pH 8.0. At high pH, the Michaelis constant for the soluble isozyme was larger than that for the mitochondrial isozyme. Gel filtration indicated that the molecular weights of the two major isozymes were approximately equal and equal to pig heart mitochondrial-malic dehydrogenase. A second mitochondrial isozyme appeared to be twice as large as the other two. In its properties, the second mitochondrial form appeared to correspond to the major mitochondrial isozyme. It is considered very significant that the properties of corn root soluble- and mitochondrial-malic dehydrogenase are similar to avian and mammalian forms.

Crassulacean Acid Metabolism in the Strangler Clusia rosea Jacq.

Observations of malic acid fluctuation, leaf anatomy, and stable carbon isotopic composition showed that the epiphytic strangler Clusia rosea, growing on Saint John, U.S. Virgin Islands, has crassulacean acid metabolism. This hemiepiphyte may be the only woody dicotyledonous tree species among the many thousands of flowering species in the 30 or more plant families that shows this type of metabolism. The finding has implications with respect to water balance during the process whereby Clusia rosea establishes itself as a tree, since crassulacean acid metabolism is a photosynthetic adaptation to water-stressed environments.

Irrigation magnifies CAM-photosynthesis in Opuntia basilaris (Cactaceae)

SummaryMeasurements of acid metabolism and gas exchange were carried out four times during a year to assess the relative importance of temperature and the accompanying seasonal change to the carbon metabolism of Opuntia basilaris Engelm. & Bigel. plants growing in situ under irrigated and natural (control) conditions. Our experiments showed that this cactus did not change its pattern of carbon assimilation when continuously irrigated under field conditions. Non-irrigated cacti had maximum acid accumulation after periods of precipitation. Maximum acid accumulation in irrigated cacti occurred when there was a large difference between day/night temperatures (i.e., 16°C), and when nighttime temperatures were moderate (14C). Irrigated cacti had greater duration of stomatal opening and lower resistance to 14CO2 uptake. When temperatures were low, daytime stomatal resistance to 14CO2 uptake decreased (to 20–40 s cm-1), but never to the level of the nocturnal resistances (5–10 s cm-1). During periods of drought, nonirrigated cycti changed to a pattern in which organic acids fluctuated. Irrigated cacti continued to have 14CO2 uptake when nighttime temperatures were as high as 33°C. 13C/12C isotope composition ratios, determined after two years of irrigation, were near -12‰ in irrigated and non-irrigated plants. Therefore, under conditions of continual irrigation, seasonal and temperature changes affected the degree of dark CO2 fixation and acid metabolism, but these cacti did not change from CAM to CO2 fixation in the light.

Activation of plant p-enolpyruvate carboxylases by glucose-6-phosphate: A particular role in crassulacean acid metabolism

Abstract P-enolpyruvate carboxylase prepared from leaves of several plants is activated by m M concentrations of glucose-6-phosphate. At 2 m M glucose-6-phosphate the activation was due to the lowering of K 0.5 for P-enolpyruvate, without appreciable effects on the maximum velocity. Glucose-6-phosphate activation of P-enolpyruvate carboxylase purified from leaves of Crassulacean plants reverses the substrate level inhibition of this enzyme by malic acid. The role of activation and inhibition is discussed in relation to the control of malic acid synthesis in Crassulacean leaves.

Ozone effects on plant cell permeability

The effect of ozone on plant cell permeability was examined. Pinto bean plants (Phaseolus vulgaris) were exposed to 0.4 ppm ozone for one hr and discs cut from primary leaves were allowed to take up /sup 14/C-UL-d-glucose or /sup 14/C-UL-2-deoxy-d-glucose. Uptake of 2-deoxy-d-glucose one hr after exposure appeared lower in exposed tissue than controls, while 24 hr after exposure uptake of both sugars by exposed tissue was three times that of controls. When uptake in one hr was examined at various times after exposure up to twenty hr it was found that permeability did not increase until several hr after the end of exposure. Cursory examination of the internal distribution of /sup 14/C label showed that the increase in permeability was not a function of increased metabolic activity since no differences were seen between exposed and control tissue, except in fractions representing cell wall material. The same percentage of /sup 14/CO/sub 2/ was released by exposed and control tissue even though uptake of glucose was greater in exposed tissue. These data are interpreted to mean that the primary targets for ozone are cellular membranes, resulting in alterations in membrane permeability.

NADP-specific malate dehydrogenase of green spinach leaf tissue

Abstract An NADP-specific malate dehydrogenase was partially purified from spinach leaf tissue by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration. The protein appears to be localized in the stroma of intact chloroplasts. For activity, there is a requirement for a reagent such as dithiothreitol (DTT). Almost complete inactivation occurs after desalting by gel filtration or exhaustive dialysis. Reactivation occurs by incubation in 5 n m DTT. The completely activated protein elutes from a calibrated Sephadex G-200 column with an apparent molecular weight of 110,000. An inactivated preparation elutes as three distinct peaks with estimated molecular weights of 29,000, 42,000, and 75,000. Apparent Kms computed from hyperbolic kinetic data were: oxaloacetate = 0.03 m m , NADPH = 0.07 m m , malate = 11.0 m m , and NADP = 0.04 m m . The data are of physiological interest since it appears that green spinach leaf tissue has MDH activity catalyzing OAA ⇌ MAL in all major subcellular compartments; viz., NAD specific activity in microbodies, mitochondria, and in the cytosol, and NADP specific activity in the chloroplasts.