Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Effects of mefluidide and dicamba on in vitro growth and embryogenesis ofDactylis glomerata (orchard grass)

The effects of N-(2,4-dimethyl-5-(((trifluoromethyl)sulfonyl)amino)phenyl)acetamide (mefluidide) and 3,6-dichloro-o-anisic acid (dicamba) on in vitro growth and somatic embryogenesis ofDactylis glomerata L. (orchard grass) were studied using suspension cultures and explanted leaf bases. All experiments employed modified Schenk and Hildebrandt medium amended with concentrations of dicamba ranging from 15 to 120 M (SH-15 to SH-120) and of mefluidide ranging from 1 to 100 M. SH medium without either growth regulator was used for embryo germination. Embyro production in suspension cultures with SH-30 medium plus 3 g/L casein hydrolysate was significantly reduced by 1 M mefluidide. Only 15% of these embryos germinated and produced plants compared to 84% from controls. Growth, as measured by dry weight, was significantly reduced by 50 or 100 M mefluidide. The number of embryos formed on leaf sections was significantly reduced by 20 or 25 M mefluidide. Embryos that formed with 10 M or more mefluidide were callused on both SH-15 and SH-30 media. Shoot formation was inhibited from individual embryos and embryo/callus masses that developed on either SH-15 or SH-30 medium containing 5 M or more mefluidide. Radicle emergence was significantly reduced with 10 M mefluidide regardless of 15 or 30 M dicamba. Histological examination revealed that mefluidide inhibited both shoot and root meristem development with shoot development being the more sensitive. Inhibition of both was independent of dicamba concentrations. Shoot formation was also reduced from embryos that had developed on SH-30 medium without mefluidide when transferred to medium containing mefluidide without dicamba.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.     

Fluorescence studies of normal and sickle beta apohemoglobin self-association

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle apohemoglobins has been studied in 0.05 M potassium phosphate buffer,pH 7.5, at 5°C over a protein concentration range from 1 to 50M. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle apohemoglobins, and were found to be 5.6 and 2.4M, respectively, thus demonstrating distinct self-association properties of _A and _S apohemoglobins.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

A meta-analysis of frontalis EMG levels with biofeedback and alternative procedures

The lack of comparative reviews of the efficacy of EMG frontalis biofeedback versus alternative procedures for reduction of muscle tension prompted the present meta-analytic treatment of literature previously concluded to be equivocal. Twenty studies comparing EMG frontalis biofeedback with other tension-reduction procedures produced a total of 68 separate effect sizes suitable for meta-analysis. Differences between clinical and normal samples were nonsignificant, and data analyses revealed that EMG frontalis biofeedback was significantly superior to control (p<.05) but that alternative forms of muscle relaxation, while effective, did not reach statistical significance.     

Probable male heterogamety in the deep-sea fish Bathylagus wesethi (Teleostei: Bathylagidae)

A presumed XY chromosome pair is described fromt estis squashes from the mesopelagic deep-sea fish Bathylagus wesethi, whose 2N chromosome number was determined as 34-XY. Although the metacentric X-chromosome is the largest in the entire compliment, the Y is the smallest and only acrocentric element. The positive heteropycnosis of the sex elements was not easily distinguishable in the nuclei of first meiotic prophase. Tetraploid nuclei were observed in peripheral supporting cells of the testis. Males of at least two other congeners have similar karyotypes.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

The “biological ego”. From Garrod's “chemical individuality” to Burnet's “self”

Starting from the conceptual premises of Garrod, who as long ago as 1902 spoke of chemical individuality, and of Burnet (1949), who recognized as self one's own molecular antigenic structures (as opposed to the antigenic alien: the non- self), the discovery and understanding of HLA antigens and of their extraordinarily individual and differentiated polymorphisms have gained universal recognition. Transplant medicine has now dramatically stressed, within man's knowledge of himself, the characteristic of his biological uniqueness. Today man, having become aware of being a biological antigenic-molecular individuality which is unique and different from that of all of his fellow men (except for monozygotic twins), can therefore easily consider himself a true biological Ego.Abbreviations BMT bone marrow transplantation - GVHD graft versus host disease - HLA human leukocyte antigens - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction     

Neutral changes during divergent evolution of hemoglobins

Summary A comparison of the mRNAs for rabbit and human-hemoglobins shows that synonymous changes in codons have accumulated three times as rapidly as nucleotide replacements that produced changes in amino acids. This agrees with predictions based on the so-called neutral theory. In addition, seven codon changes that appear to be single-base changes (according to maximum parsimony) are actually two-base changes. This indicates that the construction of primordial sequences is of limited significance when based on inferences that assume minimum base changes for amino acid replacements.     

Activation of protein kinase C alpha is required for TPA-triggered ERK (MAPK) signaling and growth inhibition of human hepatoma cell HepG2

The signaling mechanisms for most of the antiproliferative processes are not fully understood. We have demonstrated that ERK(MAPK) signaling was involved in the induction of both p15^INK4band p16^INK4a CDK inhibitors and growth inhibition of hepatoma cell HepG2 triggered by the tumor promoter tetradecanoyl phorbol acetate (TPA). In this study, the upstream signal mechanism for TPA-induced ERK(MAPK) activation was investigated. In HepG2 cells only one of the cPKC isozymes, PKC, but not cPKCII, nPKC or aPKC was activated by TPA as demonstrated by its membrane translocation within 10–30 min and down-regulation at 24 h after TPA treatment. Pretreatment of 0.2–2.0 M Bisindolylmaleimides, an inhibitor of PKC, attenuated the TPA-induced phosphorylation of ERK, gene expressions of p15^INK4band p16^INK4a, and growth inhibition of HepG2 cell in a dose-dependent manner. Consistently, transfection of HepG2 with 1.0–3.0 M antisense (AS) PKC, but not (AS) PKCII, or nPKC oligonucleotides (ODN), for 36 h prior to TPA treatment also prevented the TPA-induced molecular and cellular effects described above. Taken together, we concluded that PKC is specifically required for TPA-induced ERK(MAPK) signaling to trigger gene expressions of p15^INK4band p16^INK4a leading to HepG2 growth inhibition.     

Excitatoren und paarungsverhalten mitteleuropäischer malachiiden (Coleopt., Malacodermata)

Ohne ZusammenfassungAlphabetisches Verzeichnis der im Text gebranchten Abkürzungen AK Anbieten der Kopfgrube () - AR-seitig außenrandseitig (auf Elytre bezogen) - EO Elytralorgan (EO-Arten = Arten mit Elytralorganen im männlichen Geschlecht) - f Flucht () - F Flucht () - FA, fa frontale Auseinandersetzung (, ) - FS, fs Fühlertrillern bzw. Frontalspiel (, ) - gk Grubenknabbern () - IR-seitig innenrandseitig (auf Elytre bezogen) - K Kopulation - KG Kopfgrube (KG-Arten = Arten mit Kopfgrube im männlichen Geschlecht) - KI Abdomenkitzeln () - KV Kopulationsversuch () - LP-Feld den weiblichen Labialpalpen korreliertes Drüsenporenfeld - MP-Feld den weiblichen Maxillarpalpen korreliertes Drüsenporenfeld - 180 Drehung des um 180° - ob Organbeißen () - ok Organknabbern () - OZ Organzuwendung () - P Prüfung der Kopulationsbereitschaft durch - RB rückwärtige Berührung durch - SLV Seitwärtslauf nach vorn () - SLH Seitwärtslauf nach hinten () - U Umrundung () - vl Vorwärtslauf () - 180 Drehung des um 180° Habilitationsschrift.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

Verapamil induced reduction of the myocardial β-adrenoceptor density in BIO 14.6 cardiomyopathic Syrian hamsters

In general, it is recognized that prolonged exposure to catecholamine leads to a reduction in the -adrenoceptor density (downregulation). However, it has been previously reported that the myocardial -adrenoceptor densities and norepinephrine levels significantly increase in the hearts of BIO 14.6 cardiomyopathic hamsters in the early stage. The mechanism of the increased -adrenoceptor density is not clearly elucidated, and it can not be excluded that this phenomenon may be a secondary effect. The purpose of this study was to assess the effect of verapamil on the density of -adrenoceptors in the heart of BIO 14.6 cardiomyopathic hamsters. The total number of -adrenoceptors in untreated BIO 14.6 hamsters was significantly higher at 90 days of age (30.4±2.2 v.s. 25.9±1.4 fmol/mg protein, p<0.05). BIO 14.6 hamsters received daily intraperitoneal injections of 5 mg/kg verapamil for 70 days, from an age of 20 days. Verapamil protected against progressive myocardial damage (total damage; 8.2±0.7 v.s. 0.4±0.2%/area, p<0.05) and the myocardial -adrenoceptor density returned to that of the normal control group (26.9±3.0 fmol/mg protein). Conversely, verapamil did not have an effect on the number of myocardial -adrenoceptors in normal golden hamsters. This study showed that verapamil protected against progressive myocardial damage and myocardial -adrenoceptor density returned to those of normal hamsters. These results suggest that an increased number of -adrenoceptors in the early stage of BIO 14.6 cardiomyopathic hamsters may be involved in the secondary pathogenesis of cardiomyopathy.     

Über das Wachstum der Pankreaszellen von Säugetieren als Monolayer Cultures

Zusammenfassung Fetale menschliche Pankreaszellen bilden sowohl spontan auf zuerst gewachsenem Zellrasen (self-aggregation) als auch in Rollröhrchen (48 rph) (rotation-mediated-aggregation) histotypische Aggregate, die A- und B-Zellen enthalten. In der Nährlösung dieser 6–17 Tage lang gewachsenen Gewebestückchen konnte, Insulin (=IRI=immunoreactive-insulin) nachgewiesen werden.

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Monolayer cultures of pancreatic tissueII. Formation of histotypic aggregates from cell suspensions of fetal human pancreas

Summary Fetal Pancreas cells of man, whether in self-aggregation or rotation-mediated-aggregation, form spontaneously histotypical aggregates containing A- and B-cells. It is possible by means of an immunological method to determine insulin (=IRI=immuno-reactive-insulin) in the nutrient medium of these 6 to 17 days old tissue aggregates.

     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.