Enzyme-linked immunosorbent assay for the activator protein specific for the enzymic hydrolysis of GM1 in urine

We have established a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of the activator protein which stimulates the enzymic hydrolysis of G_M1 (G_M1-activator) in human urine. The level of G_M1-activator in 19 normal, adult urine samples was estimated to be 370.7±33.2 ng/ml. The amounts of G_M1-activator excreted in 24 h were estimated to be between 0.28 and 1.1 mg. The coefficient of variation for this method is 4.3% for the intra-assay and 14.4% for the inter-assay. Urine samples, without purification, can be used directly for the ELISA.     

A fluorometric method for monitoring the enzymic hydrolysis of terminal galactose from GM1-ganglioside.

A fluorometric method for monitoring the enzymic hydrolysis of the terminal galactose from GM₁-ganglioside has been developed. The released galactose is oxidized with galactose dehydrogenase and NAD and the fluorescence of the product NADH measured. This method can detect as little as 0.1 nmol of galactose. β-Galactosidase from the gastropod Turbo cornutus was employed for the hydrolysis reaction. The rate of GM₁-ganglioside hydrolysis is linearly proportional to incubation time for 30 min under the assay conditions employed. In addition to galactose, the other product of hydrolysis, GM₂-ganglioside, is identified by thin-layer chromatography. This procedure provides a convenient and specific method for measuring the release of galactose from GM₁-ganglioside.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

Sequence,evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (K_a) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pK_a and a negative correlation with log D of the OHPCBs. IC₅₀ values of inhibitory OHPCBs decreased with decreasing pK_a values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pK_a and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Analysis of the mutagenic properties of the UmuDC, MucAB and RumAB proteins, using a site-specific abasic lesion

ThemucAB andrumAB loci have been shown to promote mutagenesis to a greater extent than the structurally and functionally homologousEscherichia coli umuDC operon. We have analyzed the basis of this enhanced mutagenesis by comparing the influence of these operons, relative toumuDC, on the mutagenic properties of each of two abasic sites, specifically located in a single-stranded vector. Experiments with these vectors are useful analytical tools because they provide independent estimates of the efficiency of translesion synthesis and of the relative frequencies of each type of nucleotide insertion or other kind of mutagenic event. TheumuDC, mucAB, andrumAB genes were expressed from their naturalLexA-regulated promoter on low-copy-number plasmids in isogenic strains carrying aumuDC deletion. In addition, plasmids expressing the UmuD'C, MucA'B, or RumA'B proteins were also used. Compared toumuDC, the chief effect ofmucAB was to increase the efficiency of translesion synthesis past the abasic site. The enhanced capacity ofmucAB for translesion synthesis depended about equally on an inherently greater capacity to promote this process and on a greater susceptibility of the MucA protein to proteolytic processing. The RumA protein also appeared to be more susceptible to proteolytic processing, but the inherent capacity of theRum products for translesion synthesis was no greater than that ofUmuDC. dAMP was inserted opposite one of the two abasic sites studied at a somewhat greater frequency in strains expressingrum (82%) compared to those expressingumu (72%), which might result in higher mutation frequencies inrumAB than inumuDC strains.     

Structural basis of ligand binding and release in insect pheromone-binding proteins: NMR structure of Antheraea polyphemus PBP1 at pH 4.5

The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1A, was determined at 20 degrees C. The structure consists of six alpha-helices, which are arranged in a globular fold that encapsulates a central helix alpha7 formed by the C-terminal polypeptide segment 131-142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19-54, 50-108 and 97-117, which were identified by NMR. Superposition of the ApolPBP1A structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBPA, yielded an rmsd of 1.7 A calculated for the backbone heavy-atoms N, Calpha and C' of residues 10-142. In contrast, the present ApolPBP1A structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix alpha7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.     

Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells

Summary Bacillus subtilis -amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells.Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the -amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the -lactamase of pBR322 and a thermostable -amylase. The secretion of -lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable -amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable -amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.     

Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci

The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4 mg/l of cell culture for TraC and up to 10 mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.     

Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

The hexahistidine (His₆)/nickel(II)-nitrilotriacetic acid (Ni²⁺-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His₆ proteins. Here we explore several applications of the NTA derivative, (Ni²⁺-NTA)₂-Cy3. This molecule binds our two model His₆ proteins, N-ethylmaleimide sensitive factor (NSF) and O⁶-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 10⁶ M⁻¹) and no effect on their activity. Its high specificity makes (Ni²⁺-NTA)₂-Cy3 ideal for detecting His₆ proteins in complex mixtures of other proteins, allowing (Ni²⁺-NTA)₂-Cy3 to be used as a probe in crude cell extracts and as a His₆-specific gel stain. (Ni²⁺-NTA)₂-Cy3 binding is reversible in 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (k_exchange ∼ 5 × 10⁻⁶ s⁻¹ with 0.2 μM labeled protein in the presence of 1 μM His₆ peptide). Labeling with (Ni²⁺-NTA)₂-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni²⁺-NTA)₂-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.     

The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

Isolation and some properties of soluble and membrane-bound cobalamin binding proteins of Euglena mitochondria

Cobalamin binding activity occurred in the soluble fraction (69%) and the membrane fraction (31%) of Euglena mitochondria. The mitochondrial soluble cobalamin binding protein was purified about 580-fold in a yield of 34%; the membrane-bound cobalamin binding protein was solubilized with 2 M urea and partially purified. Both purified mitochondrial cobalamin binding proteins showed low pH dependency for activity. The pH optima of the soluble and membrane-bound cobalamin binding proteins were in the vicinity of 7.0 and 6.0–8.0, respectively. The K _s values of the soluble and membrane-bound cobalamin binding proteins for cyanocobalamin were 0.3 and 0.9 nM, respectively. Neither mitochondrial cobalamin binding proteins required metal ions for activity, but the activity of the soluble and membrane-bound cobalamin binding proteins was inhibited by 1 mM Mn²⁺, 48% and 89%, respectively. Molecular weight of the soluble cobalamin binding protein was calculated to be 93,000. The physiological roles of both mitochondrial cobalamin binding proteins were discussed on the basis of their properties and location in Euglena mitochondria.Abbreviations Cbl cobalamin - Ado-Cbl 5-deoxyadenosylcobalamin - CN-Cbl cyanocobalamin - Me-Cbl methylcobalamin - OH-Cbl hydroxocobalamin - 2-AMP-Cbl 2-amino-2-methylpropanolylcobalamin     

Aphid feeding deterrency of ellagitannins, their phenolic hydrolysis products and related phenolic derivatives

Feeding deterrent activities of ellagic acid, two ellagitannins, gallic acid, pyrogallol, and several gallic acid derivatives towards three species of aphids were determined. The most sensitive species tested was Schizaphis graminum (Rondani), the least sensitive was Acyrthosiphon pisum (Harris). Myzus persicae (Sulzer) was of intermediate sensitivity. Ellagic acid (ED₅₀=15 ppm) and n-decyl gallate (ED₅₀=16 ppm) were particularly potent against S. graminum, while n-octyl gallate was the most active compound tested against A. pisum (ED₅₀=182 ppm) and M. persicae (ED₅₀=56 ppm). The ellagitannins, geraniin and pedunculagin, were active against S. graminum and M. persicae, but not against A. pisum. Methylation of the free hydroxyl groups of gallic acid resulted in a large decrease in activity, while esterification of its carboxyl group with alkyl chains of increasing length resulted in increasing activity against S. graminum. Against A. pisum and M. persicae, ellagic acid, gallic acid and 3,4,5-trimethoxybenzoic acid were inactive, whereas pyrogallol and the gallate esters were at least moderately active as feeding deterrents.

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Résumé L'examen a porté sur l'action répulsive, lors de l'alimentation de trois espèces de pucerons, de l'acide ellgique, de deux ellagitanins, de l'acide gallique, du pyrogallol et de plusieurs dérivés de l'acide gallique. Schizaphis graminum Rondani a été l'espèce la plus sensible, tandis que Acyrthosiphon pisum. Harris a été la moins sensible; la sensibilité de Myzus persicae Sulzer était intermédiaire. L'acide ellagic (ED₅₀=15 ppm) et le n-décyl gallate (ED₅₀=16 ppm) ont été particulièrement actifs contre S. graminum, tandis que le n-octyl gallate a été le produit le plus actif contre A. pisum (ED₅₀=182 ppm) et M. persicae (ED₅₀=56 ppm). Les ellagitanins, géraniine et pédunculagine ont été actifs contre S. graminum et M. persicae, mais pas contre A. pisum. La méthylation des groupes hydroxyl libres de l'acide gallique a réduit fortement l'activité, tandis qu l'estérification de son groupe carboxyl avec des chaînes alkyl de longuers croissantes a augmenté l'activité contre S. graminum. Les acides ellagique, gallique et 3,4,5-triméthoxybenzoïque ont été inactifs contre A. pisum et M. persicae, tandis que le pyrogallol et les esters de gallate on été pour le moins des répulsifs modérément actifs au cours de l'alimentation.

     

Isolation for bacteria and fungi for the hydrolysis of phthalate and terephthalate esters

Bacterial and fungal strains were isolated from enrichment cultures using diethylphthalate, diethylterephthalate, or ethylene glycol dibenzoate as sole carbon sources.Aureobacterium, Flavobacterium, andMicrococcus species were isolated from diethylphthalate enrichments;Rhodococcus andXanthomonas species were isolated from diethylterephthalate enrichments;Rhodococcus andFusarium species were isolated from ethylene glycol dibenzoate enrichments.     

Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system

The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made between the SRK₉₁₀ kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1. SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK₉₁₀ kinase domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK₉₁₀ kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Identity of the activator proteins for the enzymatic hydrolysis of sulfatide,ganglioside GM1, and globotriaosylceramide

The activator protein for the enzymatic hydrolysis of sulfatide, ganglioside G_M1, and globotriaosylceramide was purified from human kidney, brain, and urine. As far as they could be assayed, these three activities cochromatographed during all steps, indicating that they are due to the same protein. This result was corroborated by immunochemical comparison of individually purified activator preparations. In contrast, the activator for ganglioside G_M2 hydrolysis could clearly be separated from the other activities. Kinetic data were determined for the interaction of the sulfatide activator with the different glycolipids and hydrolases.