Characterization of inulosucrase and the enzymatic synthesis of inulin / 生物工程学报

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²⁺ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.

Hyper-production of levansucrase from Zymomonas mobilis KIBGE-IB14 using submerged fermentation technique

The industrial utilization of enzymes requires the high yield of enzyme production for the synthesis of polymers by microorganisms. Therefore, it is necessary to optimize different production parameters of levansucrase in order to increase its industrial applications. Zymomonas mobilis KIBGE-IB14 was considered as a promising candidate for the large scale production of levan among wide range of microorganisms. The current investigation is aimed to optimize the production parameters of levansucrase by Z mobilis KIBGE-IB14 isolated from molasses. The results indicated that bacterial growth as well as enzyme production was greatly influenced by both physical and chemical conditions. It was revealed that high enzyme titers were achieved at 30°C with pH 6.5 after 24 hours of incubation in a modified medium. Moreover, the enzyme exhibited its induction in the presence of sucrose used as a substrate. Thus, the present study demonstrated that newly isolated Z mobilis KIBGE-IB14 can be used as a plausible producer of levansucrase for industrial applications

Molecular cloning and over-expression of a fructosyltransferase from Aspergillus niger QU10 / 生物工程学报

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.

Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant / 生物工程学报

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae / Identificação de Haemonchus placei e Haemonchus contortus por PCR e por análise morfológica de parasitas adultos e larvas infectantes

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

Métodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de “primers” espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.

Expression analyses of BcUGT3 and BcUGT6, and their in vitro expression in Escherichia coli / 中国中药杂志

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.

Construction, expression and enzymatic activity analysis of AUR1 eukaryotic expression vector of Botrytis cinerea / 生物工程学报

In order to study the expression and the activity of inositol phosphorylceramide synthase (BcAUR1 gene) in Botrytis cinerea, we amplified BcAUR1 by RT-PCR from Botrytis cinerea, using the special primers with FLAG and BamH I/Xho I restriction sites. Recombinant pYES2-BcAUR1 was constructed to transform into Saccharomyces cerevisae deltayorl by LiAC. The expression of inositol phosphorylceramide (IPC) synthase and its activity were detected by Western blotting and HPLC, respectively. The results show that pYES2-BcAUR1 could express in uracil mutant deltayorl of Saccharomyces cerevisae. IPC synthase enzyme activity of pYES2-BcAUR1 transformants significantly increased and was approximately double than no-load BcAUR1 transformants. The low concentration of Aureobasidin A could inhibit growth of no-load BcAUR1 transformants, but pYES2-BcAUR1 transformants could resist fungal growth inhibition which was induced by Aureobasidin A.

Cloning, expression, identification and bioinformatics analysis of Rv3265c gene from Mycobacterium tuberculosis in Escherichia coli

OBJECTIVE@#To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications.@*METHODS@#Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed.@*RESULTS@#The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained α-helix, extended strand and random coil, 46.8%, 14.6%, 38.6%, respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein.@*CONCLUSIONS@#Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.

Characterization of the functional domain of STT3a of oligosaccharyltransferase from Dunaliella salina / 生物工程学报

To investigate the function of STT3a gene in salt adaptation and flagellar regeneration of Dunaliella salina (D. salina), a pair of degenerate primers was designed according to conserved homologous amino acid sequences of VCVFTA and DVDYVL of STT3a from Chlamydomonas, Arabidopsis thaliana and other organisms. A cDNA sequence of 1 650 bp encoding a whole functional domain of STT3a was amplified from D. salina by RT-PCR and 3' Rapid Amplification of cDNA Ends (RACE), which shared homology with Chlamydomonas (48%), Arabidopsis thaliana (50%), Homo sapiens (46%), etc. Real-time fluorescence quantitative PCR (real-time Q-PCR) demonstrated that the STT3a mRNAs from D. salina were induced by increased concentration of NaCl, and increased to 11-fold higher by 3.5 mol/L NaCl than that by 1.5 mol/L NaCl (P < 0.01). Also, STT3a mRNA of D. salina maintained at a higher level in the process of flagellar regeneration with than without experiencing deflagellar treatment. In conclusion, the findings of this study demonstrate that the high expression of the STT3a gene enhances the capability of salt adaptation and flagellar regeneration in D. salina.

Regeneration of transgenic Lespedeza thunbergii expressing levansucrase gene (Sac B) from Bacillus subtilis / 生物工程学报

The Bacillus subtilis Sac B gene with the vacuolar targeting signal sequence driven by 35S promotor was transferred into Lespedeza thunbergii by Agrobacterium mediated method. Total 62 Kan-resistant plants were obtained, of which 5 plants were proved to be transgenic plants. The transgenic plants were characterized by PCR amplification, PCR-Southern hybridization and RT-PCR. The physiological assay results showed that the transgenic plants were more tolerant to stress than the controls under the condition of 200mmol/L NaCl and 5% PEG, respectively, and that the content of soluble sugar in trnsgenic plants was significantly higher than that of controls in the period of tests (5-15 days) under salt and PEG stress.

Catalytical properties of N-glycosylated Gluconacetobacter diazotrophicus levansucrase produced in yeast

The influence of N-glycosylation on the kinetic and catalytical properties of a bacterial fructosyltransferase (LsdA) produced in Pichia pastoris was studied. The glycosylated enzyme behaved similarly to non-glycosylated LsdA when substrate specificity, fructo-oligosaccharide (FOS) production, sucrose hydrolysis or levan formation reactions were carried out under different experimental conditions. The kinetic parameters for native or yeast-expressed LsdA determined at 60ºC, condition for the highest hydrolytic activity, followed a conventional Michaelis-Menten kinetics. Synthase activity of this levansucrase increased in water-restricted environments by addition of salt or organic solvent to the reaction mixtures.

Detección de meticilino-resistencia en Staphylococcus aureus: Comparación de métodos convencionales y aglutinación con mrsa-Screen latex / Methicillin resistance detection in Staphylococcus aureus: Comparison between conventional methods and Mrsa-Screen latex agglutination technique

Staphylococcus aureus meticilino-resistente (MRSA) es un patógeno que ha emergido en las últimas cuatro décadas causando tanto infecciones nosocomiales como de la comunidad. La rápida y precisa detección de MRSA es relevante para guiar una apropiada terapia antibiótica y evitar la diseminación nosocomial de MRSA.En este trabajo se evaluó la eficiencia de métodos convencionales para la detección de meticilino-resistencia como difusión por discos, CIM en medio sólido, screening de oxacilina, y el nuevo test de aglutinación MRSA-Screen latex sobre 100 aislamientos de S. aureus, 79 mecA positivos y 21 mecA negativos. El test de aglutinación MRSA-Screen latex (Denka Seiken, Niigata, Japón) detecta la presencia de la PLP-2a, producto del gen mecA en cepas de S. aureus. La detección del gen mecA por PCR se utilizó como gold standard para comparar los resultados de los diferentes métodos. La sensibilidad y especificidad fueron 97 y 100 % para el método de difusión, 97 y 95 % para la CIM en medio sólido, 100 y 100 % para el screening de oxacilina y 100 y 100 % para MRSA-Screen latex. Todos los métodos presentaron alta sensibilidad y especificidad, pero el “MRSA-Screen latex” mostró la ventaja de poder brindar un resultado confiable, equivalente a la PCR, en sólo 15 minutos.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the “gold standard” for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100 %, agar dilution 97 and 95 %, oxacillin agar screen test 100 and 100 %, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Analysis of antimicrobial resistance of Streptococcus pneumoniae with restriction fragment length polymorphism of pbp2b gene and pulsed-field gel electrophoresis profiles among children / 中华儿科杂志

<p><b>UNLABELLED</b>Streptococcus pneumoniae is a common cause of potentially life-threatening infections such as meningitis, bacteraemia, pneumonia worldwide, for which children of preschool age are at particularly high risk. Since the late 1970s and 1980s, antibiotic resistance among pneumococci has become an emerging problem. Several multidrug-resistant clones have rapidly spread throughout the world.</p><p><b>OBJECTIVE</b>(1) To investigate the prevalence of penicillin and other antibiotics nonsusceptibility among pneumococci. (2) To analyze the correlation of pbp2b amplicon profiles with penicillin resistance. (3) To serotype 31 isolates of penicillin-resistant pneumococci by latex agglutination. (4) To analyze the chromosomal relatedness of serotype 23F and 6 isolates of penicillin-resistant pneumococci by using pulsed-field gel electrophoresis (PFGE) and characterize these isolates in molecular epidemiology.</p><p><b>METHODS</b>(1) Susceptibility was determined by using broth microdilution, E-test, and K-B disk. (2) The correlation of pbp2b amplicon profiles with penicillin resistance was assessed by restriction fragment length polymorphism (RFLP). (3) Serotyping of penicillin-resistant pneumococcal isolates was performed by using latex agglutination. (4) The properties of serotype 23F and 6 isolates of penicillin-resistant pneumococci were assessed by PFGE.</p><p><b>RESULTS</b>S. pneumoniae with increased nonsusceptibility (including intermediate strains and resistant strains) to penicillin G was 9.9% in 1997, 12.6% in 1998, 14.6% in 2000; to cefuroxime 4.2%, 1.5%, 8.2%; to cefotaxime 0.0%, 1.7%, 1.0% respectively. There were no statistically significant differences (P > 0.05). While resistance to erythromycin, trimethoprim-sulfamethoxazole and chloramphenicol increased significantly from 76.8% in 1997 to 87.4% in 2000, from 74.7% to 88.3%, and from 22.6% to 40.8%, respectively (P < 0.05). RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. Of the 31 strains of penicillin-resistant pneumococci (MICs 0.12 - 2.0 micro g/ml) studied, 6 (19.4%) strains (MICs 0.12 - 0.19 micro g/ml) were serotype 23F and 3 (9.7%) strains (MICs 0.5 - 1.5 micro g/ml) were serotype 6. There were nearly identical susceptibility to antibiotics and identical PFGE patterns in the former, and there were different susceptibility to antibiotics and different PFGE patterns in the latter. Three serotype 6 strains had different susceptibility to antibiotics and different PFGE patterns, which suggested that those strains may be scattered.</p><p><b>CONCLUSION</b>Generally beta-lactams retained their activity against S. pneumoniae in Beijing. Resistance to erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol increased drastically. RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. In 6 strains of serotype 23 F there were nearly identical susceptibility to antibiotics and identical PFGE patterns, which suggested the probability that there was a spread of serotype 23F isolates with low-level penicillin resistance in local area.</p>

Effects of ganglioside GM1 on reduction of brain edema and amelioration of cerebral metabolism after traumatic brain injury / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To observe the effects of ganglioside GM1 on reduction of brain edema and amelioration of cerebral metabolism after traumatic brain injury (TBI).</p><p><b>METHODS</b>An acute experimental closed TBI model in rats was induced by a fluid-percussion brain injury model. At five and sixty minutes after TBI, the animals were intraperitoneally injected by ganglioside GM1 (30 mg/kg) or the same volume of saline. At the 6th hour after TBI, effects of ganglioside GM1 or saline on changes of mean arterial pressure (MAP), contents of water, lactic acid (LA) and lipid peroxidation (LPO) in the injured cerebral tissues were observed.</p><p><b>RESULTS</b>After TBI, MAP decreased and contents of water, LA and LPO increased in brain injury group; however, MAP was back to normal levels and contents of water, LA and LPO decreased in ganglioside GM1 treated group, compared with those in brain injury group (P < 0.05). No significant difference between the saline treated group and the brain injury group (P > 0.05) was observed.</p><p><b>CONCLUSIONS</b>Ganglioside GM1 does have obvious neuroprotective effect on early TBI.</p>

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus.

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.

Early Screening of Oxacillin-Resistant Staphylococcus aureus and Staphylococcus epidermidis from Blood Culture

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.

Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases.

Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.