Serological studies on phenolase from spinach leaves

Antibodies were prepared against phenolase Form X, one of the electrophoretically fast-moving forms (VII-X) which are spontaneously liberated from thyl     

Intracuticular lipids of spinach leaves

The cuticular wax and cutin components of the cuticular membranes isolated from the leaves of two spinach cultivars have been determined. The membranes contain about 0·007 mg/cm² of cuticular wax which comprises monobasic acids (C₁₆–C₃₈) with hexadecanoic as the major component. The amounts of cutin are comparable with those of cuticular wax and the monomeric constituents are predominantly C₁₈ epoxy compounds. The most abundant monomer is 9,10-epoxy-18-hydroxyoctadecanoic acid (up to 63%) together with substantial amounts of 9,10,18-trihydroxyoctadecanoic acid (up to 22%). Also present are 9,10-epoxyoctadecane-1,18-dioic acid (6–7%) dihydroxyhexadecanoic acid (3–4%) and ω-hydroxymonobasic and fatty acid fractions. The tentative identification of two minor components, 18-hydroxyoxooctadecanoic and 9,10-epoxy-12,18-dihydroxyoctadecanoic acids, is also made. Although spinach membranes have a delicate structure their cutin composition is essentially similar to that of much more substantial membranes.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Effects of denaturing agents on spinach nitrate reductase

The effects of various denaturing agents (temperature, pH, urea, guanidine hydrochloride and sodium dodecyl sulphate) on the 3 enzymic activities of th     

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I_a-I_b and II_a-II_b). The fractions with low electrophoretic migration rate—I_b and II_b—are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I_a and II_a-originate from the corresponding fractions I_b and II_b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.     

Association by 2,3-dihydroxybenzaldehyde of monomeric phenolase in spinach chloroplasts

In the presence of 5 mM 2,3-dihydroxybenzaldehyde, the monomeric phenolase (MW 36000) of spinach chloroplasts is completely converted to its dimer within 6 hr without significant change in activity. The aldehyde at concentrations higher than 0.25 mM could bring about this conversion after 18 hr treatment. The association of the two monomers becomes tighter with increasing concentration of the aldehyde. The dimer gave rise to a higher MW protein after freezing briefly. Several mono- and dihydroxybenzaldehydes, 2,3-dihydroxybenzoic acid, and o-vanillin did not produce the dimer.     

Multiplicity of spinach root phenolase and its monophenolase activity

Soluble phenolase of spinach roots is present in multiple forms, none of which is electrophoretically identical with those detected in the chloroplasts     

The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1_c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10⁵ and 3.7 × 10⁵). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1_c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1_m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10⁵. Electrophoresis separated 1_m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10⁵ and 1.28 × 10⁵). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1_c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1_m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1_c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1_m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1_c but not 1_m. Electrophoresis after dissociation indicates that 1_c and 1_m are both enzyme complexes. After dissociation, the 1_c complex but not that of 1_m could be reassociated. The ribonuclease of the 1_m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1_c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1_m complex being concerned with RNA catabolism.     

Volatile components of sugar beet leaves

Extracts of both young and old sugar beet plants were obtained using a modified Likens and Nickerson apparatus. Constituents were identified by GC/MS, and using selected ion monitoring it was shown that the previously determined phenylacetonitrile was probably not of glucosinolate origin. Some unsaturated aldehydes, alcohols and derivatives (enzymic lipid degradation products) were formed to greater extents by the younger leaves, but otherwise such quantitative differences were relatively few and generally random. An interesting range of chlorinated compounds was obtained only from the older plants; a pesticide origin is suggested.     

Activities and multiplicity of phenolase from spinach chloroplasts during leaf ageing

Phenolase activity in spinach leaves homogenates depends on the stage of development of leaves and on the kind of homogenization procedure. Under constant experimental conditions it is low in non-senescent leaves. With the onset of senescence there is a 15–20-fold increase in soluble activity in the supernatants of broken chloroplasts as well as an increase in activation of latent phenolase in fractions containing thylakoids. This rise in activity is due to an increase in particular multiple forms, differing for supernatants and membrane sediments. Phenolase from spinach lacks monophenolase and laccase activities.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Molecular weight and amino acid composition of purified spinach beet phenolase

The purification of spinach beet phenolase has been modified to include equilibration of the crude macerate with 0.5% cetyl-trimethylammonium bromide a     

Activation of latent phenolase from spinach chloroplasts by ageing and by frost

Activation of latent phenolase by freezing and thawing occurs in both thylakoid sediments and membrane washings from spinach chloroplasts, while ageing and digitonin treatment activates membrane-bound enzyme only. Disc clectrophoresis reveals that frost converts a soluble, latent phenolase to an active form after its release from the thylakoid membrane. Ageing of membranes containing latent phenolase results in direct liberation of other active forms. There are further active, soluble forms, which are exclusively found in the chloroplast stroma fraction.     

Activation and inhibition of spinach ribulose 1,5-bisphosphate carboxylase by 2-phosphoglycolate

Ribulose 1,5-bisphosphate carboxylase when activated by preincubation with 1 mM bicarbonate and 10 mM magnesium chloride can be further activated ca 20–500% by incubating with 2.5 mM phosphoglycolate depending upon the pH of the preincubation medium. The activation effects were seen only under specific preincubation conditions. The activation by phosphoglycolate was a slow reaction requiring ca 15 min for maximal effect. Even though magnesium was essential for phosphoglycolate activation, concentrations higher than 15 mM progressively inhibited the activation of the enzyme by phosphoglycolate. When added directly to the reaction mixture, phosphoglycolate was a potent inhibitor of the carboxylase activity. Even under preincubating conditions, phosphoglycolate showed slight inhibitory effect at 0.1 mM and activation was observed at concentrations higher than 0.5 mM. The K_A value for phosphoglycolate was 2.8 mM.     

Inhibition by oxalates of spinach chloroplast phenolase in unfrozen and frozen states

Spinach chloroplast phenolase was inhibited by oxalic acid and its salts. Complete inhibitions were induced instantly in the acidic region (e.g. by 1 and 5 mM oxalate at pH 5 and 5.5, respectively), and in the neutral region pre-incubation of the enzyme with oxalates could also lead to complete loss of activity. The inhibition mode was non-competitive for phenol substrate with K_i of 0.9 mM pH 6.8. Reduction of enzyme activity in a crude extract of chloroplasts induced by freezing at neutral pH was due to the presence of ammonium oxalate. With 0.5 mM oxalate, the inhibition attained 75% under frozen conditions, whilst no inhibition could be detected in the enzyme which had not been frozen. Free oxalic acid and K⁺ and Na⁺ salts also caused freezing inhibition. Glyoxylic and oxamic acids acted as inhibitors with less efficiency. With a pure mushroom tyrosinase (phenolase), essentially the identical results were obtained using the same conditions.     

Relation between photosynthetic and phenolase activities in spinach chloroplasts

During O°-storage of class I chloroplasts from spinach leaves, activation of phenolase strongly correlates with the inactivation of photosynthetic re     

Magnesium deficiency results in accumulation of carbohydrates and amino acids in source and sink leaves of spinach

Accumulation of assimilates in source leaves of magnesium‐deficient plants is a well‐known feature. We had wished to determine whether metabolite concentrations in sink leaves and roots are affected by magnesium nutrition. Eight‐week‐old spinach plants were supplied either with a complete nutrient solution (control plants) or with one lacking Mg (deficient plants) for 12 days. Shoot and root fresh weights and dry weights were lower in deficient than in control plants. Mg concentrations in deficient plants were 11% of controls in source leaves, 12% in sink leaves and 26% in roots, respectively. As compared with controls, increases were found in starch and amino acids in source leaves and in sucrose, hexoses, starch and amino acids in sink leaves, whereas they were only slightly enhanced in roots. In phloem sap of magnesium‐deficient and control plants no differences in sucrose and amino acid concentrations were found. To prove that sink leaves were the importing organs they were shaded, which did not alter the response to magnesium deficiency as compared with that without shading. Since in the shaded sink leaves the photosynthetic production of metabolites could be excluded, those carbohydrates and amino acids that accumulated in the sink leaves of the deficient plants must have been imported from the source leaves. It is concluded that in magnesium‐deficient spinach plants the growth of sink leaves and roots was not limited by carbohydrate or amino acid supply. It is proposed that the accumulation of assimilates in the source leaves of Mg‐deficient plants results from a lack of utilization of assimilates in the sink leaves.     

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.