Aubrey W. Naylor

Calibration and use of a Clark-type oxygen electrode from 5 to 45°C☆

Abstract A calibration procedure for a Clark-type oxygen electrode over a wide range of temperatures is described. The autoxidation of duroquinol (2,3,5,6-tetramethyl-1,4-benzenediol) was used to verify the electrodes ability to accurately sense the total amount of dissolved O 2 in an aqueous buffer. Electrode response time was measured by using oxygenated ethanol to deliver a rapid increase in O 2 concentration to the reaction medium. An oxygen-producing system (spinach thylakoids) was utilized to test the range of O 2 -evolution rates able to be sensed. It was concluded that a Clark-type oxygen electrode has the absolute sensitivity, rapidity, and range necessary to accurately track rates of O 2 production or consumption from 5 to 45°C.

A simple electrophoretic procedure for separation of RNA on mixed agarose—acrylamide gel columns

A simple procedure is described for the preparation of mixed agarose—acrylamide gels for the separation of high molecular weight species of RNA by a modification of the method of Peacock and Dingman1. A method for the measurement of radioactivity in fractionated gels is described which permits tritium determination with efficiencies as high as 40%.

A method for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves

Abstract A method is described for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves of two higher plant species. Sodium dodecyl sulfate (1%) and 25 mM magnesium ions are required to inhibit ribonuclease action during RNA purification by phenol deproteinization. The ethanol-precipitated RNA product, including 23s chloroplast ribosomal RNA, is completely stable during electrophoresis in the absence of magnesium ions, even in the presence of EDTA. The in vivo mole fraction of chloroplast ribosomes relative to cytoplasmic ribosomes is estimated. Bentonite is shown to cause preferential losses of chloroplast RNA during extraction.

Purine and pyrimidine nucleotide inhibition of carbamyl phosphate synthetase from pea seedlings.

Carbamyl phosphate synthetase from Alaska pea seedlings is subject to inhibition by several pyrimidine and purine nuclotides. It is similar in some respects to the E.coli enzyme. Both have similar Ki values for UMP, the most potent inhibitor, and both show partial reversal of UMP inhibition by L-ornithine. However, the pea enzyme is inhibited by AMP and ADP, while the E.coli enzyme is stimulated by these compounds. The role of nucleotides and ornithine in strongly influencing the activity of carbamyl phosphate synthetase has obvious potential regulatory significance in the operation of the ornithine cycle and in pyrimidine nucleotide synthesis.

Effects of light on phenylalanine ammonia-lyase activity in dark-grown Zea mays (L.) seedlings

Abstract The effects of continuous white light on phenylalanine ammonia-lyase (PAL) activity in Zea mays L. roots and mesocotyls was studied. White light increased enzyme specific activity in both roots and mesocotyls. Anthocyanin accumulation in roots and mesocotyls in continuous white light up to 48 h correlated with the kinetics of light-induced PAL specific activity, anthocyanin biosynthesis lagging several hours behind light-induced PAL specific activity. When illumination was given for varying time periods followed by a dark period which ended 48 h after the beginning of the illumination period, kinetics of anthocyanin biosynthesis based on time in the light corresponded well with the light-induced PAL specific activity, indicating a possible causal relationship. Level of enzyme activity per unit of fresh weight was increased by light in both root and mesocotyl, however, total enzyme activity per mesocotyl was not changed by light as was total enzyme activity per root.

Ontogenetic Study of Urease in Jack Beans, Canavalia ensiformis (L.) DC.

Urease occurs in all parts of the jack bean (Canavalia ensiformis [L.] DC.) plant. The floral parts contain 1/6-1/10 as much urease as the vegetative shoot apices and green leaves. The embryo and the seedling, excluding cotyledons containing about 1% of the total units, showed a constant amount of urease up to 10 days following the initiation of germination. Shoot apices contained 1-2 units of urease per 25 apices for a period of about 7 weeks and possibly thereafter. Studies on cotyledons of germinating seedlings showed the existence of three phases in urease activity. For the first few days, the urease content remained constant, and the specific activity increased somewhat; the second phase was characterized by a declining urease content but increasing specific activity; and in the last phase both the amount of urease and its specific activity declined. Dark-grown seedlings showed the same pattern except that the first phase was somewhat prolonged. The peak of proteolytic activity of the cotyledonary extracts was observed in the first phase around 6-7 days after germination began. Possibly, changes in the specific activity of urease may be a reflection of the prevailing proteolytic activity. Studies on cotyledons of maturing seeds showed that the increase in urease units per gram of dry wt increased with increasing dry wt of the cotyledons, and the rate of synthesis was very high in the 6-7 week post-fertilization period. A difference of 7-10 days in the age of the seeds from pods harvested in this period was reflected in the urease content. In the last few weeks of maturation of seeds, however, the rate of urease synthesis was slow, and at this stage the differences in age did not show up in the urease measurements. Small-sized fully mature seeds may possess double the specific activity of the large-sized seeds on a per-gramprotein basis. Probably, in the later part of seed maturation proteins other than urease are synthesized in greater amounts in the seeds attaining a large size than in the small ones.

Culture of Pine Root Callus and the Use of Pinus clausa Callus in Preliminary Metabolic Studies

1. Root-callus cultures of five species of southern pines-pond (Pinus serotina Michx.), longleaf (Pinus palustris Mill.), slash (Pinus elliottii Englm.), loblolly (Pinus taeda L.), and sand (Pinus clausa [Chapm.] Vasey)-were established on agar containing modified Hellers solution and were maintained by serial transfer. Sand pine root callus was selected for the studies reported here. 2. Manometric experiments with respiratory inhibitors revealed inhibition of respiration by cyanide, iodoacetic acid, and fluoride, but not by fluoroacetate or malonic acid, in buffer solutions of pH 5.4. 3. Polyphenol oxidase, peroxidase, and catalase, but not cytochrome oxidase, activities could be demonstrated in tissue extracts. 4. Radioactive glucose, formate, pyruvate, glutamate, citrate, and succinate feedings demonstrated that the utilization of these compounds by sand pine callus cultures was not radically different from that of other higher plants. 5. The initial products accumulating label in the dark from C14O2, detected in our chromatographic system, were malate and aspartate. Subsequent distribution of activity was into members of the citric acid cycle and transamination products of oxalacetate and α-ketoglutaric acid.

STUDIES ON THE ORNITHINE CYCLE IN ROOTS AND CALLUS TISSUES OF PINUS SEROTINA AND PINUS CLAUSA

1. Isolated roots of pond pine (Pinus serotina Michx.) and root-callus tissue cultures of pond pine and sand pine (Pinus clausa [Chapm.] Vasey) were used in studies of the ornithine-urea cycle. 2. Ornithine-2-C14 was surmised to be utilized by the plant material in two main ways: (a) via citrulline and arginine and (b) via glutamic semialdehyde to glutamic acid and proline. 3. Both roots and callus tissue incorporated C14O2 in darkness into citrulline as well as into the usual products of dark CO2 fixation. The amount of C14O2 incorporated into citrulline was increased either by growing the plant materials on a medium containing ornithine before supplying HC14O- 3, or by incubating the tissues simultaneously with ornithine and HC14O- 3. 4. Sodium formate-C14 was also utilized partly via citrulline. Oxidation of formate to CO2 in the tissue may account for some of the observed results. 5. Pond pine was more active than sand pine in metabolizing ornithine-2-C14, NaHC14O3, and sodium formate-C14 via citrulline and arginine. 6. A complete cyclic process, leading to urea synthesis, has not been demonstrated in the tissues studied.

STUDIES ON NITROGEN FIXATION BY ALNUS CRISPA

Root nodules of Alnus crispa (Ait.) Pursh were shown to possess a symbiotic nitrogen-fixing organism. The reduction of acetylene to ethylene, as measured by gas chromatography, was used to determine the presence of the nitrogen-fixing system. Ethylene production was measured at 5.1 μmoles/g excised nodule · hr for both field and greenhouse plants. The nodules were found to consist of short nubs usually clustered in masses up to 4 cm in diam. Microscopic examination of nodules revealed some cortical cells fully packed with spherical endophyte cells. The outer cortex and radiating arms of cells in the inner cortex remained uninfected. Nodules examined during the winter were found to be shrunken, with a random distribution of endophyte cells. Soil nitrogen measurements indicated that nitrogen fixation activity by A. crispa does not lead to an increase in soil nitrogen above levels in adjacent areas.

Light effects on phenylalanine ammonia-lyase substrate levels and turnover rates in maize seedlings

Abstract If extracted phenylalanine ammonia-lyase (PAL) activity reflects in vivo activity, there should be a correlation between light-induced changes in extracted PAL activity and light-induced changes in PAL substrate turnover rates. Light-induced increases in levels of extracted PAL activity from dark- grown maize seedlings exposed to continuous white light are accompanied by corresponding decreases in free pools of phenylalanine and tyrosine, the two substrates of PAL in maize. Evidence is presented which indicates that these changes are not the result of changes in protein synthesis. The light-induced change in turnover rate of phenylalanine, is shown to be strongly correlated with light-induced PAL activity in roots of maize seedlings, indicating extracted PAL activity reflects in vivo activity.