Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea.

Nascent pectin and glucuronoarabinoxylan, synthesised in vitro by membrane-bound enzymes from etiolated pea (Pisum sativum L.) epicotyls, were found to bind to pea xyloglucan in a pH-dependent manner. The binding was maximum at low pH (3-4), and decreased to almost zero at pH 6. The binding was probably non-covalent and reached saturation within 5 min. Removal of the fucose residues of xyloglucan decreased the degree of binding. Removal by protease of the proteins attached to nascent pectin and glucuronoarabinoxylan greatly reduced the maximum binding and abolished the pH-dependence. The observed binding may be of considerable significance in the process of cell-wall assembly and in the control of cell extension.

Binding of nascent glucuronoxylan to the cell walls of pea seedlings.

Glucuronoxylan synthesised in vitro by membrane-bound enzymes from etiolated pea epicotyls was found to bind to isolated cell walls from the same tissue in a pH-dependant manner. The binding was maximum at pH 3.5-4.0, and decreased to zero at pH 6. The bound glucuronoxylan could be dissociated from the cell walls by washing at pH 6, and the binding appeared to be non-covalent. Extraction experiments indicated that the glucuronoxylan was binding to hemicellulose in the cell-wall. The observed binding may be significant in the process of cell-wall assembly in vivo.

Differential distribution of a glucuronyltransferase, involved in glucuronoxylan synthesis, within the Golgi apparatus of pea (Pisum sativum var. Alaska).

The subcellular location of a glucuronyltransferase (GT) involved in glucuronoxylan synthesis in pea (Pisum sativum) has been investigated. Most of the GT activity was found in the Golgi fraction, but activity was also detected in the plasma-membrane fraction. Separation of Golgi membranes on a shallow continuous sucrose density gradient resulted in three distinct subfractions, with GT activity being confined to Golgi membranes of a density similar to that of smooth endoplasmic reticulum. The differential distribution of GT within the Golgi stack indicates that glucuronoxylan synthesis occurs in specific cisternae and that there is functional compartmentalization of the Golgi with respect to hemicellulose biosynthesis.

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.

The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.

Cytochemical localization of 5'-nucleotidase in the frog (Rana pipiens) retina. A histochemical and cytochemical study.

Localization of 5'-nucleotidase in the frog retina was investigated using histochemical and cytochemical techniques. Light-microscopic observations revealed the presence of this enzyme in the inner retinal layers (the nerve fiber layer, ganglion cell layer, and inner plexiform layer). Ultrastructural investigations revealed that the enzyme activity is associated with the plasma membranes of the Müller cell processes, whereas the Müller cell processes present in the outer retinal layers did not demonstrate any detectable enzyme activity. This observation would appear to confirm our previous findings, that 5'-nucleotidase is an ectoenzyme, but its distribution in frog retina differs from that in rodents and it is only present in the inner layers of the retina. The prominent localization of 5'-nucleotidase on the glial plasma membrane may be viewed in the context of the widely accepted interaction between neurones and glial cells. Since nucleotides do not penetrate the plasma membrane, a mechanism to produce membrane-permeable adenosine, important for neuronal function, is postulated. It is known that 5'-nucleotidase produces adenosine by hydrolyzing adenosine 5'-monophosphate (5'-AMP). Therefore one would expect that the glial membrane-bound enzyme can accomplish the final step in this mechanism by producing the adenosine in the extracellular spaces.

The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone.

It has been suggested that pectic polysaccharides (or oligosaccharides cleaved from them) are liberated from the cell wall upon wounding of leaf tissue, and that they act as long-distance hormones evoking a defence response in neighbouring uninjured leaves (P.D. bishop et al. 1981, Proc. Natl. Acad. Sci. USA 78, 3536-3540, and cited literature). We have tested this hypothesis by infiltration of radioactive pectic fragments (rhamnogalacturonans and homogalacturonans of degrec of polymerisation down to 6) into wounds on tomato leaves. No radioactivity was exported from the treated leaf. [(14)C]Sucrose, applied in the same way, was effectively translocated, probably via the phloem. We suggest that pectic substances are not themselves long-distance wound hormones. The possibility remains that pectic substances, solubilised on wounding, act in the immediate vicinity of the wound to stimulate the dispatch of a second messenger, which would be the long-distance wound hormone.

The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro.

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.

Isolation and characterization of membranes from the cells of maize root tips.

A discontinuous sucrose density gradient was used to separate membrane fractions from a homogenate of maize root tips. Endoplasmic reticulum-, Golgi apparatus-, plasma membrane- and mitochondria-rich fractions were identified by their enzymic characteristics and by their appearance under the electron microscope. Maize roots were incubated in vivo with D-[U-14C]glucose, [Me-14C]choline chloride and diazotized [U-3H]sulphanilic acid. The pattern of incorporation of radioactivity into the various membrane fractions was investigated. Analyses of the polypeptide chains of the membrane fractions by SDS-polyacrylamide gel electrophoresis showed that the mitochondria-rich fraction had a different pattern of polypeptides from that of the other membrane fractions. The results are discussed in relation to the hypothesis of endomembrane flow and differentiation.

Measurement and characteristics of fusion of isolated membrane fractions from maize root tips.

Maize root tips were incubated in vivo with radioactive glucose, choline or diazotized sulphanilic acid. Membrane fractions were prepared from radioactive and non-radioactive roots. The transfer of radioactivity between mixed membrane fractions has enabled a quantitative system to be developed to study in vitro membrane fusion between Golgi apparatus and plasma membrane-rich fractions. Membrane fusion was found to be dependent on time, temperature, Mn2+ and Ca2+. Mn2+ was as effective as Ca2+, other divalent cations had a moderate or no effect. The effect of various substances, including inhibitors of microtubular and microfilament assembly and blocking reagents for sulphydryl group on membrane fusion has been investigated. The process appears to be dependent on the membrane proteins or glycoproteins; trypsinization of mixed membranes prior to the addition of Ca2+ inhibited significantly the fusion process. SDS-polyacrylamide gel electrophoresis of trypsin-treated membranes revealed the selective loss of one particular polypeptide which could play a role in membrane fusion.