A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases

Paquet, Marie‐Eve; Moscarello, Mario A.

doi:10.1042/bj2180745

Cited by 17 publications

(3 citation statements)

References 20 publications

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“…Bovine milk ␤4-galactosyltransferase showed higher a K m for UDP-Gal, in agreement with previous studies (37,(55)(56)(57)(58), and the measured K m for GlcNAc was similar to that determined in some studies (59,60), but 5-10-fold higher compared with other studies (55-58). As shown in Fig.…”

Section: Identification and Cloning Of Human ␤4gal-t2supporting

confidence: 81%

A Family of Human β4-Galactosyltransferases

Almeida¹,

Amado²,

David³

et al. 1997

Journal of Biological Chemistry

169

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BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase, designated ␤4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of ␤4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with ␤4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of ␤4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases, thus designated ␤4Gal-T2 and ␤4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with ␤4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple ␤4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to ␤4Gal-T1.During the last decade, more than 40 mammalian glycosyltransferases have been cloned and characterized (1, 2). The initial strategy for cloning glycosyltransferases was cumbersome purification of labile enzyme proteins followed by screening of cDNA libraries with antibodies or DNA probes based on amino acid sequence information (3-11). The introduction of transfection cloning by Lowe and co-workers (12) resulted in a marked increase in the cloning of novel glycosyltransferase genes (13-17). A third successful approach has taken advantage of conserved sequences in glycosyltransferases that share donor and/or acceptor substrates. Thus, searches for novel members of homologous glycosyltransferase gene families utilizing conserved sequence motifs for RT-PCR 1 cloning with degenerate primers have resulted in the identification and cloning of a number of novel genes (18,19).One part of the human genome project is the establishment of a data base of expressed sequence tags (ESTs), which currently has over 700,000 unique sequences. ESTs represent short 5Ј-and 3Ј-sequences (200 -500 bp) of cDNA clones from a large variety of human and animal organs (20). The EST data base is now estimated to contain sequence information from more than half the human genes; it therefore provides a unique source for identifying novel members of homologous gene families (21). The EST data base has recently been successfully utilized in searches for novel glycosyltransferase genes of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, where several novel members of this homologous gene family have been iso...

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Section: Identification and Cloning Of Human ␤4gal-t2supporting

confidence: 81%

A Family of Human β4-Galactosyltransferases

Almeida¹,

Amado²,

David³

et al. 1997

Journal of Biological Chemistry

169

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“…The enzyme was retained on a a-lactalbumin-Sepharose column in the pres ence of N-acetylglucosamine in the buffer and was released from the column when Nacetylglucosamine was omitted from the elu tion buffer. This affinity chromatographic step resulted in an about 2,000-fold purifica tion of the enzyme -a result close to that of Paquet and Moscarello [14] who have puri fied the enzyme from rat serum about 2,300-fold by this one-step purification procedure. As seen in table I, partial purification of the calf serum galactosyltransférase did not im prove the Co2+ activity of the enzyme.…”

Section: Partially Purified Calf Serum Galactosyltransférasesupporting

confidence: 52%

Co^2+ Is Able to Substitute for Mn^2+ in Some Exogenous and Endogenous Galactosyltransférase Reactions

Gmeiner¹

1988

Enzyme

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The ability of Co^2+ to substitute for Mn^2+ in exogenous and endogenous galactosyltransférase reactions was tested. Exogenous transfer was measured towards different high and low molecular weight galactose acceptors using galactosyltransférase from the following sources: crude serum, the serum enzyme partially purified by affinity chromatography and a pure enzyme preparation from milk. Endogenous transfer was estimated in preparations from human urinary bladder tumor cells and from rat liver microsomal fractions. The results show that Co^2+ is able to substitute for Mn^2+ in some exogenous and endogenous galactosyltransférase reactions. This ability seems to depend on the molecular structure of the galactose acceptor as well as on the nature of the enzyme.

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“…Bivalent metal ions are an absolute requirement for the activity of galactosyltransferase (EC 2.4.1.22), a Golgilocated enzyme that attaches galactosyl fl1-4 residues in glycoproteins and, in the presence of ac-lactalbumin, in lactose. Maximum activity is elicited by Mn2+ ions, although their optimum concentration has variously been reported as 40-50 mm (Babad & Hassid, 1964;Khatra et al, 1974), 10-13 mM (Spiro & Spiro, 1968;Babad & Hassid, 1966) or only 4-5 mm (Morrison & Ebner, 1971a,b;Paquet & Moscarello, 1984). However, recognizing that such concentrations of Mn2+ are unlikely to occur in vivo, Powell & Brew (1976) reinvestigated the galactosyltransferase purified from bovine milk and gave evidence for two distinct metalion activation sites.…”

Section: Introductionmentioning

confidence: 99%

Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins

Navaratnam¹,

Virk²,

Ward³

et al. 1986

Biochemical Journal

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Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.

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A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases

Cited by 17 publications

References 20 publications

A Family of Human β4-Galactosyltransferases

A Family of Human β4-Galactosyltransferases

Co^2+ Is Able to Substitute for Mn^2+ in Some Exogenous and Endogenous Galactosyltransférase Reactions

Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins

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