Sucrose Density Gradient Separation Research Articles

Annexin A2 expression and partners during epithelial cell differentiation.

The members of the annexin family of calcium- and phospholipid-binding proteins participate in different cellular processes. Annexin A2 binds to S100A10, forming a functional heterotetrameric protein that has been involved in many cellular functions, such as exocytosis, endocytosis, cell junction formation, and actin cytoskeleton dynamics. Herein, we studied annexin A2 cellular movements and looked for its partners during epithelial cell differentiation. By using immunofluorescence, mass spectrometry (MS), and western blot analyses after S100A10 affinity column separation, we identified several annexin A2-S100A10 partner candidates. The association of putative annexin A2-S100A10 partner candidates obtained by MS after column affinity was validated by immunofluorescence and sucrose density gradient separation. The results show that three proteins are clearly associated with annexin A2: E-cadherin, actin, and caveolin 1. Overall, the data show that annexin A2 can associate with molecular complexes containing actin, caveolin 1, and flotillin 2 before epithelial differentiation and with complexes containing E-cadherin, actin, and caveolin 1, but not flotillin 2 after cell differentiation. The results indicate that actin, caveolin 1, and E-cadherin are the principal protein partners of annexin A2 in epithelial cells and that the serine phosphorylation of the N-terminal domain does not play an essential role during epithelial cell differentiation.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

Heterodimeric capping protein from Arabidopsis is a membrane-associated, actin-binding protein.

The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.

Caveolin‐1 Supports the P2Y2 Receptor Signaling

Damage to the CNS cells’ can cause a differential spatiotemporal release of multiple factors into the extracellular space. Among such factors, nucleotides, and their interaction with the P2Y2 nucleotide receptors (P2Y2Rs) have gained prominence as putative modulators of gliotic responses after CNS injury. Yet, the molecular mechanisms underlying these interactions and responses remain to be explored. Discontinuous sucrose density gradient separation of 1321N1 cells (expressing recombinant hP2Y2R) homogenates revealed co‐fractionation of P2Y2Rs in cav‐1 (+) membrane‐raft fractions. Likewise, confocal microscopy revealed that a significant percent of P2Y2Rs co‐localized with cav‐1 in its sub‐cellular distribution. Blocking cav‐1 expression in hP2Y2‐1321N1 cells elicited abnormal intracellular Ca2+ mobilization responses and uncharacteristic patterns of AKT, ERK1/2 and p38 phosphorylation levels when stimulated with nucleotide agonists, as determined by microfluorometric calcium imaging analyses and immunoblotting, respectively. Our findings suggest that P2Y2Rs reside in membrane caveolae of non‐stimulated 1321N1 cells and that this sub‐cellular compartment may couple its downstream signaling machinery. We hypothesize that P2Y2Rs’ signaling cascades are linked to their expression in cav‐1 (+) micro‐domains. NAM is supported by the NIGMS‐NIH award number R25GM061838 at the UPR‐MSC.

Protein Kinase A Subunit α Catalytic and A Kinase Anchoring Protein 79 in Human Placental Mitochondria

Components of protein phosphorylation signalling systems have been discovered in mitochondria and it has been proposed that these molecules modulate processes including oxidative phosphorylation, apoptosis and steroidogenesis. We used electrophoresis and Western blots probed with specific antibodies to protein kinase A α catalytic subunit (PKAα Cat) and A kinase anchoring protein of approximately 79 kDa molecular weight (AKAP79) to demonstrate the presence of these two proteins in human placental mitochondria. Heavy mitochondria characteristic of cytotrophoblast were separated from light mitochondria characteristic of syncytiotrophoblast by centrifugation. PKAα Cat and AKAP79 were present in both heavy and light mitochondria with no significant difference in concentration. Sucrose density gradient separation of submitochondrial fractions indicated PKAα Cat is located predominantly in the outer membrane whereas AKAP79 is present mainly in the contact site fractions. These data indicate that PKAα Cat is present in the cytoplasm, nucleus and mitochondria of placental cells. AKAP79 is also present in human placental mitochondria but there may be anchoring proteins other than AKAP79 responsible for fixing PKA to the outer membrane. PKA may play roles in mitochondrial protein phosphorylation systems in both cytotrophoblast and syncytiotrophoblast.

CD38/ADP-ribosyl cyclase in the rat sublingual gland: Subcellular localization under resting and saliva-secreting conditions

CD38 is a 42–45kDa transmembrane glycoprotein that exhibits ADP-ribosyl cyclase enzyme activity. In the rat, we have previously reported strong ADP-ribosyl cyclase activity in the sublingual salivary gland (Masuda W. and Noguchi T. Biochem. Biophys. Res. Commun. (2000) 270, 469–472). Here, we have examined the specific localization of CD38/ADP-ribosyl cyclase activity in this gland and whether that localization changes upon saliva-secretary stimulation. Under resting conditions, CD38/ADP-ribosyl cyclase activity in the post-nuclear fraction of SLG homogenates was separated into two major peaks by sucrose density gradient centrifugation. The first peak included the plasma membrane proteins Na+/K+ ATPase and aquaporin 5, while the second peak included mucous secretory protein mucin and vesicle-associated membrane protein 2. When rats were subjected to the muscarinic agonist pilocarpine, the CD38/ADP-ribosyl cyclase activity disappeared from the second peak, as did mucin and vesicle-associated membrane protein 2. Pre-treatment of rats with the muscarinic antagonist atropine before pilocarpine administration, or adrenergic stimulation with isoproterenol, the sucrose density gradient separation profiles were same as that seen under resting condition. Using an immunofluorescent strategy, we observed the preferential localization of CD38 in the basolateral plasma membrane and intracellular granule-like membrane in sublingual acinar cells under resting conditions.

Evidence for secretion of Cu,Zn superoxide dismutase via exosomes from a cell model of amyotrophic lateral sclerosis

A familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), is caused by dominant mutations in the cytosolic Cu,Zn superoxide dismutase (SOD1). There has been evidence for secretion of SOD1, by an unknown mechanism. In this work stable mouse motor neuron-like NSC-34 cells overexpressing human SOD1 wild-type hSOD1(wt) (NSC-34/hSOD1(wt)) and mutant hSOD1(G93A) (NSC-34/hSOD1(G93A)) have been used as an ALS cell model. SOD1 was found to be secreted in association with a membrane fraction that pelleted at 100,000xg. Sucrose density gradient separation of this fraction showed that wild-type and mutant SOD1 were found between 0.5 and 1.16M sucrose and co-localized with the exosomal marker CD9. Therefore, SOD1 secretion occurred via exosomes. p115 a cytosolic and Golgi apparatus (GA) protein involved in vesicle tethering was also found in exosomes, contrary to the endoplasmic reticulum protein calnexin. SOD1 secretion mediated by exosomes could explain cell-to-cell transfer of mutant toxicity.

A Cholesterol-independent Membrane Microdomain Serves as a Functional Counter-receptor for E-selectin at the Colo201 Cell Surface and Initiates Signalling on E-selectin Binding

The present study demonstrates that the functional counter-receptors for E-selectin at the cell surface of Colo201 human colon cancer cells are localized in detergent-insoluble membrane microdomains (DIM). Following isolation of counter-receptors from whole cell lysates using E-selectin-coupled magnetic beads followed by sucrose density gradient separation, both sialyl Lewis a (SLe(a))- and sialyl Lewis x (SLe(x))-carrying glycoproteins which had bound to the E-selectin-beads were distributed in detergent-soluble fractions as well as DIM. In contrast, following isolation of counter-receptors directly from the cell surface, SLe(a)-carrying glycoproteins which had bound to E-selectin-beads at the cell surface were localized only in DIM, together with a Src family kinase, Lyn, while SLe(x)-carrying glycoproteins were not detected in any fraction. The counter-receptors were distributed in a diffuse pattern on the cell surface but clustered following E-selectin binding, leading to the subsequent phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of the cells with methyl-beta-cyclodextrin, a cholesterol-depleting drug, had little effect on either the association of SLe(a)-carrying glycoproteins and Lyn with the domain or ERK phosphorylation. Thus, the functional counter-receptors and Lyn are co-localized in a cholesterol-independent microdomain and create a physiological domain ('glycosynapse') at the cell surface that initiates signalling in cancer cells upon binding to E-selectin.

Pigment Binding, Fluorescence Properties, and Oligomerization Behavior of Lhca5, a Novel Light-harvesting Protein

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.

Assembly of the D1 Precursor in Monomeric Photosystem II Reaction Center Precomplexes Precedes Chlorophyll a–Triggered Accumulation of Reaction Center II in Barley Etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.

Characterization of a 14 kDa component with low expression in a unique Nod+ Fix- Bradyrhizobium japonicum

A pronase-sensitive, 14 kDa component of bacteroids of Bradyrhizobium japonicum I-110ARS was identified and characterized using monoclonal antibodies. This component was weakly synthesized or was missing in bacteroids of a unique Nod- Fix- mutant, ARS2525. Both the 14 kDa component and poly-β-hydroxybutyrate (PHB) were located in the same protein peak after sucrose density gradient separation of lysed bacteroids. Thirty-five-day-old bacteroids of B. japonicum I-110ARS contained up to 10 times more PHB than B. japonicum ARS2525 bacteroids. Immunocytochemistry of ultra-thin nodule sections showed that the component was associated with PHB granules in the bacteroids, possibly with the limiting membrane around the granules. This observation implies that the component may be similar to other storage lipid body proteins. Whether low synthesis of the 14 kDa component or low PHB content was a cause, a consequence or related to the Fix- phenotype was not established.

ATP-insensitive interaction of the amino-terminal region of the beta heavy chain of dynein with microtubules.

The ATP-insensitive microtubule-binding site of dynein has been investigated by limited proteolysis of sea urchin sperm flagellar axonemes. Mild tryptic digestion cleaved the dynein beta chain at either of two principal cleavage sites, generating two sets of complementary peptides. Inclusion of ATP in the digestion medium had no effect on the generation of these primary fragments. Sucrose density gradient separation and immunostaining with monoclonal antibodies against epitopes on the beta chain showed that extraction of the digested axonemes with 1-3 mM ATP solubilizes the peptides located at the carboxy-terminal end of the original heavy chain. The solubilization of the peptides containing the amino end required the presence of 0.6 M NaCl and was not affected by ATP. While the outer arm dynein is in situ on the axoneme, the N-terminal 125-kDa domain of the beta chain was not digested by trypsin, whereas in soluble dynein this domain becomes rapidly degraded. These data suggest that the N-terminal domain of the beta chain is involved in its ATP-insensitive attachment to microtubules and support the hypothesis that the N-terminal 125-kDa peptide corresponds to the flexible tail of the dynein molecule seen in electron micrographs.

Multiple estrogen binding sites in the uterus: Stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [ 3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [ 3H]IA enantiomers (IA- R and IA- S) and [ 3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [ 3H]ligand; DES, IA-Rac, IA- R and IA- S. IA- S enantiomer ( K d =0.67) binds to the estrogen receptor with the same affinity as DES ( K d =0.71) and four times higher affinity than IA R( K d =2.56). The number of binding sites for IA- S is approximately the same as estradiol, DES and IA-Rac while IA- R binds far fewer sites than the other ligands. Saturation binding assays indicated that [ 3H]DES and [ 3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [ 3H]DES and [ 3H]IA compounds bound to the 8S form of the receptor, the same as E 2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displayed by the addition of excess unlabeled E 2 and DES. Further characterization of the 4S component is described.

Differential diagnosis of Taenia saginata and Taenia solium with DNA probes

A size selected genomic DNA library was constructed using DNA extracted from Taenia saginata. The DNA was digested using the restriction enzyme EcoR1 under star conditions and the 2-4 kbase fraction, selected following sucrose density-gradient separation, was cloned in the bacteriophage lambda gt 10. A panel of cestode DNAs including Taenia saginata, Taenia solium, Taenia taeniaeformis, Taenia crassiceps, Echinococcus granulosus and DNAs of bovine, porcine and human origin were used in conjunction with hybridization analysis to identify two recombinant bacteriophages. The first probe, designated HDP1, reacted specifically with T. saginata DNA. The second, designated HDP2, reacted with DNA from both T. saginata and T. solium but not the other DNA samples and thus provided a general reagent for positive identification of fragments of Taenia spp. proglottides of human faecal origin. If used in conjunction the two DNA probes allow positive identification of T. saginata. In the clinical situation it is important to be able to distinguish T. saginata and T. solium infections and DNA probes such as these may be useful in such differentiation.

Influence of sputum IgA and elastase on tracheal cell bacterial adherence.

Bacterial adherence is an important pathogenetic mechanism for airway colonization, but the influence of airway proteins on this phenomenon is largely unknown. We measured tracheal cell bacterial binding in 13 subjects with chronic tracheostomy and related these results to measurements of sputum IgA, elastase activity, and total protein from the same subjects. Tracheal cell adherence was related directly to sputum elastase activity (r = 0.61, p = 0.02); and elastase activity, primarily a serine protease, was higher in subjects colonized by Pseudomonas aeruginosa than in those without this finding (p = 0.02). Sputum levels of IgA/mg protein were related inversely to tracheal cell adherence (r = 0.64, p = 0.02). Sputum IgA concentrations, in turn, were affected by host nutritional status and airway elastase activity. Evidence that elastase can degrade sputum IgA was provided by an inverse relationship observed between these 2 proteins (r = 0.56, p = 0.04) and by in vitro mixing experiments showing fragmentation of IgA by purified neutrophil elastase. In addition, sucrose density gradient separation indicated IgA fragmentation to have occurred in vivo. These data suggest that, once adherence leads to airway colonization, the resulting inflammatory response may foster microbial growth by an elastase-dependent IgA cleavage and hence enhanced tracheal cell adherence.

Subcellular location of enzymes of ammonia assimilation and asparagine synthesis in root nodules of Lupinus albus L.

The subcellular localization of enzymes involved in ammonia assimilation and synthesis of asparagine in the host cells of the central, N 2-fixing zone of nodules of Lupinus albus L. has been examined using discontinuous and continuous sucrose density gradient separation of the soluble, plant organelle and bacteroid components of nodule extracts. Both glutamine synthetase (EC 6.3.1.2) and asparagine synthetase (EC 6.3.5.4) activities were cytosolic while significant NADH: glutamate synthase (EC 2.6.1.53) and aspartate aminotransferase (EC 2.6.1.1) activities were recovered together with starch-containing plastids. NAD: glutamate oxidoreductase (glutamate dehydrogenase; EC 1.4.1.2) was associated almost completely with intact mitochondria. The distribution of enzymes indicates complex subcellular organization of pathways of asparagine synthesis.

Properties of Substantially Chlorophyll-Free Pea Leaf Mitochondria Prepared by Sucrose Density Gradient Separation

Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, alpha-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca(2+), whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O(2) uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.

Demonstration and characterisation of a testicular receptor for 1,25-dihydroxycholecalciferol in the rat.

Defective luteinizing‐hormone‐mediated cAMP generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25‐dihydroxycholecalciferol (1,25‐dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25‐dihydroxycalciol receptor in the rat testis.Testes of non‐rachitic, rachitic or acutely uremic male Sprague‐Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25‐[3H]‐dihydroxycalciol (with or without 200‐fold molar excess of unlabeled 1,25‐dihydroxycalciol, subjected to 5–20% sucrose density‐gradient separation (centrifugation with 255000xg, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule in testicular cytosol with the properties of the intestinal 1,25‐dihydroxycalciol receptor. The binding macromolecule migrated at 3.5 S and could clearly be distinguished from the 6‐S tissue‐binding site for 25‐hydroxycholecalciferol. The 1,25‐dihydroxycalciol receptor was present in non‐rachitic, rachitic and acutely uremic rats.Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25‐dihydroxycalciol ≫ 25‐hydroxycholecalciferol > 1 α‐hydroxycholecalciferol > 24(R),25‐dihydroxycholecalciferol), low capacity (Nmax= 82 fmol/mg protein) and an equilibrium constant of Kd= 2.6 × 10−10 M. The receptor is a protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N‐ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25‐dihydroxycalciol. Binding of 1,25‐dihydroxycalciol protects the receptor against inactivation with N‐ethylmaleimide.

Regulatory cells in human bone marrow: Suppression of an in vitro primary antibody response

Human bone marrow (BMC) contains regulatory cells that can suppress the in vitro primary PFC response of normal allogeneic spleen or tonsillar cells and autologous peripheral blood cells. Suppression is dependent upon the dose of BMC added, but is not due to cell crowding nor to excessive cytotoxicity, and requires the presence of viable, metabolically active BMC. BMC are maximally inhibitory when added during the first 24 hr of culture and do not cause an induced shift in the kinetics of the response. Thus, suppression reflects inhibition of early inductive events in the antibody response. The target of suppression is the non-T cell, with either polyclonal activator or Ag being required for maximal suppression. DNA synthesis of normal tonsillar cells is not inhibited by BMC. Characterization of the human bone marrow-suppressor cell has shown it to be radiosensitive, E-rosette negative, Fc receptor positive, and to reside in the large, weakly adherent cell population after velocity sedimentation and in the lymphocyte-depleted fraction after sucrose density gradient separation. Pretreatment of the bone marrow-suppressor cell with anti-human thymocyte serum does not abrogate suppression. We speculate on a possible physiologic role for this cell.

The location of the enzymes of mmonia assimilation in root nodules of Phaseolus vulgaris L.

The location of the enzymes potentially involved in ammonia assimilation in nitrogen fixing root nodules of Phaseolus vulgaris has been examined by sucrose density gradient separation of the bacteroids from plant organelles. Glutamine synthetase is almost entirely present in the plant cytoplasm, glutamate dehydrogenase (GDH) is in the mitochondria. The results also show that there is little evidence for the presence of significant proportions of the total glutamine synthetase and glutamate synthase activity being present in the bacteriod. In contrast however, by far the greater proportion of the total nodule GDH activity was found in the bacteroid. The problems encountered by previous workers in assaying and localising glutamate synthase and GDH in legume root nodules are discussed.