Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

Inhibition by light of growth and Golgi-localized glucan synthetase in the maize mesocotyl

When dark-grown maize (Zea mays L.) seedlings were exposed to red light (R), Golgi-localized glucan synthetase activity in the mesocotyl began to decrease within 1 h, and fell by approx. 70% in 12 h. The response required at least 10^-2 mol m^-2 R and saturated at 100 mol m^-2. Far-red light (FR) alone inhibited glucan synthetase, and FR reversed the inhibition by R back to the level caused by FR alone. Density gradient fractionation indicated that of the major membrane markers only the Golgi-localized glucan-synthetase activity was affected by R. Golgi-localized latent inosine-diphosphatase activity was unaffected. The kinetics of the response, the photon fluence dependence, and the reversibility by FR all correlated with the inhibition by light of elongation of the mesocotyl, indicating that light inhibits growth and glucan synthetase activity by a similar mechanism.Abbreviations FR far-red light - GS glucan synthetase - IAA indole-3-acetic acid - R red light     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

Action of red light on indole-3-acetic-acid status and growth in coleoptiles of etiolated maize seedlings

Brief irradiation of intact etiolated seedlings of maize (Zea mays L.) with red light (R; 30 W cm^-2, 10 min) reduces the amounts of diffusible and free (solvent-extractable) indole-3-acetic acid (IAA) obtainable from excised coleoptile tips. The effect is transient, the lowest level (30% of the dark control) occurring at about 3 h after irradiation. The free-IAA content of the whole coleoptile and the diffusible-IAA yield from the base of the same organ are similarly reduced, whereas the conjugated-IAA content of the coleoptile is not affected. These results support the view that R inhibits the production of IAA at the coleoptile tip. It is further shown that R inhibits biosynthesis of [³H]IAA from [³H]tryptophan supplied to the coleoptile tip. The shapes of the fluence-response curves obtained for the reduction of the diffusible-IAA yield by R and far-red light (FR) indicate the participation of two photoreactive systems. One has thresholds at 10^-3 W s cm² of R, five orders of magnitude less than the minimum required for the appearance of spectrophotometrically measurable far-red-absorbing form of phytochrome (Pfr) in vivo, and 10^-1 W s cm^-2 of FR; its response is linear to the logarithm of fluence exceeding five orders of magnitude. The other system is seen above 10² W s cm^-2 as an increase in the slope of the fluenceresponse curve; its response is FR reversible and related to the Pfr level of total photoreversible phytochrome. Both systems inhibit biosynthesis of IAA from tryptophan. Elongation of the coleoptile is stimulated by R; the stimulation is most apparent in the apical region, and is saturated with a fluence at which bo detectable pfr is formed. Farred light can also saturate this response. Since the endogenous IAA concentration in the coleoptile appears not to be in the inhibitory range, it is concluded that the stimulation of coleoptile elongation is not the result of changes in free-IAA levels.Abbreviations FR far-red light - IAA indole-3-acetic acid - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light     

Expression of genes for plastid membrane proteins in barley under intermittent light conditions

Barley plants grown under intermittent light show a plastid membrane composition intermediate between those of etioplasts and chloroplasts. In particular protochlorophyll reductase disappears from the membranes whereas the 32000 protein, coded for by chloroplast DNA, becomes integrated into the membranes. The light-harvesting chlorophyll a/b protein does not accumulate within the membranes even after 11 d of development, while the corresponding mRNA can already be observed after 4 d and is translated under in vivo conditions.Abbreviations LHCP light-harvesting chlorophyll a/b protein - IL intermittent light - LD light-dark (12-h day) - EGTA ethyleneglycol-bis(oxy-ethylenenitrile)tetraacetic acid     

Inhibition of elongation and H+ and K+ transport by the phosphodiesterase inhibitor aminophylline in plant tissue

Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H⁺ and K⁺ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.Abbreviations cAMP adenosine 3:5-cyclic monophosphate - FC fusicoccin - IAA indole-3-acetic acid - MES 2-N-morpholinoethanesulfonic acid - PDE cyclic nucleotide phosphodiesterase     

A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles

A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%.     

The electrolytic preparation of bioactive radioiodinated parathyroid hormone of high specific activity.

A technique for the preparation of microgram quantities of bovine parathyroid hormone (bPTH) labeled with carrier-free ¹²⁵I to a specific activity of 1300 Ci/mmol is described. A restructured and simplified apparatus was used for electrolytic iodination, making it feasible to use reaction volumes of 100 to 200 ml. The miniaturized setup requires only a small platinum crucible connected via an agar-KCl salt bridge to a saturated KCl solution, a battery to drive the reaction, and a voltmeter to monitor the potential difference between the reference-saturated KCl solution (via a calomel electrode) and the platinum crucible. The [¹²⁵I]-labeled bPTH elutes as a single species when chromatographed on a Biogel P-10 column equilibrated in 3 m guanidine HCl-2.3 m formic acid, and it retains full biologic activity when bioassayed in vivo. It is evident that bPTH labeled to a high specific activity with ¹²⁵I does not suffer in regard to its biological potency.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.