Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin

PI(4,5)P₂ localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca²⁺-triggered vesicle exocytosis, but the impact of local PI(4,5)P₂ hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca²⁺-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P₂ hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca²⁺-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca²⁺ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K⁺ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K⁺ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K⁺ but not with 56 mm K⁺ activated PLCη2 to catalyze PI(4,5)P₂ hydrolysis. The decrease in PI(4,5)P₂ promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca²⁺-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P₂ for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca²⁺-dependent regulator of F-actin dynamics and vesicle trafficking.     

Uricase and allantoinase in glyoxysomes

In fat-degrading tissues of seedlings of seven different plant species examined, uricase activity (urate:O₂ oxidoreductase, EC 1.7.33) was associated with particulate fractions. After equilibrium density centrifugation on sucrose density gradients the enzyme activity was recovered in the glyoxysomal band (density: 1.25 grams per cubic centimeter). Allantoinase is also present in glyoxysomes but, equally, in the proplastid region (density: 1.22 grams per cubic centimeter). Xanthine oxidase, xanthine dehydrogenase, allantoicase, and urease were not detected in glyoxysomes from castor bean endosperm. Uricase in these particles shows its maximal activity at pH 8.9. The apparent K_m is 7.4 μm. Urate concentrations greater than 120 μm as well as certain other purine compounds inhibit the enzyme. Cyanide at a concentration of 10 μm is a potent inhibitor. 2,6-Dichlorophenolindophenol did not substitute for oxygen as electron acceptor.     

Isolation of active mitochondria from tomato fruit

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl₂, 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 × g for 15 minutes. The supernatant is then centrifuged at 11,000 × g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl₂, 10 mm tris-HCl, 10 mm KH₂PO₄ and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and `Honey Dew' melon.     

Requirement for extraction of polyribosomes from barley tissue

The isolation of barley (Hordeum vulgare L.) polyribosomes, showing minimal degradation effects of endogenous RNase, required a buffer adjusted to pH 8.0 and containing 0.40 m KCl in addition to common extraction components. The extracted polyribosomes were characterized in sucrose gradients by their conversion to monosomes when incubated with pancreatic RNase and by their dependence on adequate amounts of Mg²⁺ during extraction and analysis. Factors which contributed to polyribosome stability were evaluated by the relative sedimentation rates of aggregates in sucrose gradients. Tissue extraction at KCl concentrations less than 0.40 m and below pH 8.0 resulted in an appearance of larger amounts of ribosomes in the less dense region of the sucrose gradient after centrifugation. The addition of 10 mm dithiothreitol was partially effective in preventing the loss of higher polymerized states of polyribosomes at KCl concentrations below 0.40 m. Extractions conducted at KCl concentrations greater than 0.40 m and at pH 8.0 reduced the amount of ribosomes obtained from the tissue. The monosome portion of the polyribosomal profile was partially dissociated into subunits when the tissue was extracted in 0.60 m KCl. A similar effect on monosomes was obtained when polyribosomes were incubated with cycloheximide and 0.40 m KCl, a result not observed by use of a combination of 0.10 m KCl and the drug or 0.40 m KCl alone.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H⁺-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-α-d-glucopyranoside, phenyl-α-d-thioglucopyranoside, and phenyl-α-d-4-deoxythioglucopyranoside (PDTGP; K_i = 67, 180, and 327 micromolar, respectively). The K_m for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-α-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-α-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-α-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-α-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K_i values ranging from 600 to 800 micromolar, and phenyl-α-d-thiomannopyranoside, phenyl-β-d-glucopyranoside, and phenylethyl-β-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-α-d-thioglucopyranoside and PDTGP, were transported.     

Polyribosomes from peas: an improved method for their isolation in the absence of ribonuclease inhibitors

Profiles of polyribosomes were obtained from etiolated stem segments of Pisum sativum L. var. Alaska isolated in various buffers. Tissue homogenized in a medium containing 0.2 m tris-HCl, pH 8.5, 0.2 m sucrose, 30 mm MgCl₂, and 60 mm KCl yielded polyribosomes exhibiting far less degradation than tissue homogenized in conventional media containing tris-HCl at lower ionic strength and pH. A further decrease in degradation was found when polyribosomes were sedimented through a sucrose pad buffered at pH 8.5 prior to centrifugation. Increased separation was obtained using heavy (125-500 mg/ml), linear sucrose gradients. Using these techniques, messenger RNA species bearing up to 12 ribosomes (dodecamers) were resolved, with messenger RNA chains bearing 9 ribosomes (nonamers) being the most abundant (having the highest absorption peak). The data presented suggest that buffer of high ionic strength and high pH was more effective in preventing degradation of polyribosomes than was diethyl pyrocarbonate and, furthermore, that ratios involving large polyribosomes (hexamers and larger) were more accurate indices of degradation than were ratios involving total polyribosomes.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na⁺ gradient (extravesicular Na⁺ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na⁺-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K⁺ or Li⁺ are substituted for Na⁺, or if SO₄²⁻ replaces Cl⁻ as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K⁺ efflux occurs concomitant with Na⁺ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.     

Kinetics of Vibrio cholerae sialidase action on gangliosidic substrates at different supramolecular-organizational levels

G_d1a, G_d1b and G_t1b gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with G_m1 ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of G_d1a and 3′-sialosyl-lactose were employed. The apparent V_max. of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside G_m1, and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside G_d1a, the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on G_d1a lipid-free carbohydrate. With each of the used gangliosides the apparent K_m values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3′-sialosyl-lactose and on G_d1a lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg-dependent enzyme

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl₂, and 1 mM Pb(NO₃)₂. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Separation and partial characterization of multiple ribonucleic Acid polymerases from soybean hypocotyl

Two major peaks of RNA polymerase activity have been routinely separated by diethylaminoethyl cellulose chromatography following solubilization from soybean (Glycine max L. var. Wayne) chromatin. The relative amounts of these two peaks depend upon the manner in which the chromatin is purified. Pelleting the chromatin through dense sucrose solutions results in not only a loss of total solubilized RNA polymerase activity but also a selective loss of the α-amanitin-sensitive form of the enzyme. Peak I elutes from a diethylaminoethyl cellulose column at a KCl concentration of approximately 0.27 m, is insensitive to α-amanitin and rifamycin, and has Mg²⁺ + Mn²⁺ optima of 5 mm and 1.25 mm, respectively. The enzyme is inhibited by KCl concentrations of about 0.03 m or greater. Peak II elutes from the column at a KCl concentration of approximately 0.35 m, is sensitive to α-amanitin, insensitive to rifamycin, and has Mg²⁺ + Mn²⁺ optima of 2 mm and 1.0 mm, respectively. Activity is inhibited by KCl concentrations of about 0.06 m or greater. Both enzymes prefer denatured calf thymus DNA, but peak II exhibits a stronger preference.     

Inactivation of maize phosphoenolpyruvate carboxylase by urea

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.     

Evidence for active Phloem loading in the minor veins of sugar beet

Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mm uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied ¹⁴C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO₂ production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mm dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mm ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mm dinitrophenol, 4 mm ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mm dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocation in the source leaf.     

Activation of polyphenol oxidase of chloroplasts

Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or —18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.     

Modification of apparent phytochrome synthesis in pisum by inhibitors and growth regulators

The repeated exposure of Pisum (pea) plants to red light brings into operation an apparent synthesis of phytochrome which is not observed in material kept in the dark. This process shows some temperature compensation but has an optimum at 26°; it is irreversibly inhibited by 10⁻⁴ m cycloheximide and 10 μg/ml actinomycin D. It is also inhibited by the auxins indoleacetic acid, naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid at 10⁻⁴ m but in these cases the inhibition is completely reversed when the auxin is washed out of the tissue. Antiauxins 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxy isobutyric acid, while strongly inhibiting growth have little effect on apparent synthesis. Other growth regulators and the precursor of tetrapyrrole synthesis, δ-aminolevulinic acid, have no consistent effect on the process, but 3 × 10⁻⁴ m cobalt (II) nitrate is inhibitory. The capacity for apparent synthesis decreases as the cells approach maturity. The results may be explained by either de novo synthesis of phytochrome, or by a transformation process resembling in some respects the dark reversion of Pfr to Pr. The physiological role of apparent synthesis is suggested.