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PubMed1983

Substrate specificity of the form of cytochrome P-450 catalyzing the 4-hydroxylation of debrisoquine in man.

In the present study we have investigated the substrate specificity of the form of cytochrome P-450 catalyzing the 4-hydroxylation of debrisoquine in man by analyzing the kinetics of inhibition of this activity by potential alternative substrates for the enzyme. All three compounds for which there is good in vivo evidence for an association between their metabolism and the debrisoquine oxidation polymorphism (viz., sparteine, guanoxan and phenformin) were potent competitive inhibitors of the reaction. The Ki for sparteine was 85 microM, for guanoxan it was 30 microM, and for phenformin it was 205 microM. Two compounds, acetanilide and antipyrine, for which the in vivo evidence was against an association between their metabolism and that of debrisoquine, were weak, noncompetitive inhibitors of debrisoquine 4-hydroxylase activity. The Ki values were 1.23 mM and 19.3 mM, respectively. Two additional compounds, tolbutamide and amylobarbitone, for which the in vivo evidence was also against an association between their metabolism and the debrisoquine oxidation polymorphism, did not appreciably inhibit the reaction. In fact, amylobarbitone caused a slight stimulation of activity. It is concluded that debrisoquine 4-hydroxylase is a specific form of cytochrome P-450 with a well-defined substrate specificity. Furthermore, it should be possible to identify compounds that might be subject to an oxidation polymorphism prior to the exposure of any subjects to the compound.

Antipyrine↗
PubMed1999

Activity and substrate specificity of the murine STK2 Serine/Threonine kinase that is structurally related to the mitotic regulator protein NIMA of Aspergillus nidulans.

We isolated a murine STK2 (mSTK2) cDNA that is homologous to murine Nek1 serine/threonine kinase, a family member related to the cell cycle regulator kinase NIMA of Aspergillus nidulans. Structural comparison demonstrated that the kinase domain of mSTK2 is highly similar to NIMA/Nek family but the C-terminal region is not similar to any proteins except for human STK2 (hSTK2). Similarly to Nek1, mSTK2 is expressed ubiquitously among various organs and is upregulated in the testis. The expression and localization of mSTK2 are not associated with the cell cycle progression of mitogen-activated lymphocyte and DNA-transfected fibroblast. The substrate specificity of mSTK2 is similar to NIMA, but the phosphorylation is observed exclusively upon threonine residues rather than serine. The mSTK2 is shown to be a new member of the NIMA/Nek family with similar substrate specificity, which might participate in a different role from NIMA kinase involved in the cell cycle regulation.

Amino Acid Sequence↗
PubMed1962

Observations on the substrate specificity of DOPA decarboxylase from ox adrenal medulla, human phaeochromocytoma and human argentaffinoma.

The substrate specificity of the dopa decarboxylases of ox adrenal medulla, human phaeochromocytoma and human argentaffinoma have been studied. The enzymes from all three tissues decarboxylated dopa, metatyrosine, orthotyrosine and 5-hydroxytryptophan. Dopa was decarboxylated most rapidly and 5-hydroxytryptophan least rapidly by the enzyme from all three tissues. Competition experiments indicate that all four substrates are decarboxylated by the one enzyme. Attempts to demonstrate decarboxylation of [C(14)]-tyrosine, [C(14)]-tryptophan or [C(14)]-histidine by these enzyme preparations were unsuccessful.

5-Hydroxytryptophan↗
PubMed2000

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Amino Acid Sequence↗
PubMed1993

A comparative analysis of the kinetic mechanism and peptide substrate specificity of human and Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways, to structural and nonstructural proteins encoded by retroviruses, hepadnaviruses, picornaviruses, and reoviruses, as well as to several transforming tyrosine kinases. hNmt has been purified 230-fold from an erythroleukemia cell line. The monomeric enzyme has no associated methionyl aminopeptidase activity. To determine the enzyme's kinetic mechanism, we examined the effect of covariation of subsaturating concentrations of myristoyl-CoA and peptide substrate on initial velocity. Double-reciprocal plots excluded a double displacement (ping-pong) mechanism. Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against peptide substrates. Together these results are consistent with a sequential ordered mechanism where, in a typical catalytic cycle, myristoyl-CoA binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products. This kinetic mechanism is identical to that described for Saccharomyces cerevisiae N-myristoyl-transferase (Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylation in these two species. Comparative studies of the peptide substrate specificities of hNmt and Nmt1p using a panel of 12 octapeptides revealed distinct differences in their tolerance for amino acid substitutions at positions 3, 4, 7, and 8 of parental peptides derived from the amino-terminal sequences of known N-myristoyl-proteins. This finding contrasts with our recent observation that the acyl-CoA substrate specificities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop species-specific enzyme inhibitors.

Acyl Coenzyme A↗
PubMed2006

Substrate specificity of chitinases from two species of fish, greenling, Hexagrammos otakii, and common mackerel, Scomber japonicus, and the insect, tobacco hornworm, Manduca sexta.

Three chitinase isozymes, HoChiA, HoChiB, and HoChiC, were purified from the stomach of the greenling, Hexagrammos otakii, by ammonium sulfate fractionation, followed by column chromatography on Chitopearl Basic BL-03 and CM-Toyopearl 650S. The molecular masses and pIs of HoChiA, HoChiB, and HoChiC are 62 kDa and pH 5.7, 51 kDa and pH 7.6, and 47 kDa and pH 8.8, respectively. Substrate specificities of these chitinases were compared with those of another fish stomach chitinase from the common mackerel, Scomber japonicus (SjChi), as well as two from the tobacco hornworm, Manduca sexta (MsChi535 and MsChi386). The efficiency parameters, kcat/Km, toward glycolchitin for HoChiA and SjChi were larger than those for HoChiB and HoChiC. The relative activities of HoChiA and SjChi toward various forms of chitin were as follows: shrimp shell or crab shell alpha-chitin > beta-chitin >> silkworm cuticle alpha-chitin. On the other hand, the relative activities of HoChiB and HoChiC were beta-chitin >> silkworm alpha-chitin > shrimp and crab alpha-chitin. MsChi535 preferred silkworm alpha-chitin to shrimp and crab alpha-chitins, and no activity was observed toward beta-chitin. MsChi386, which lacked the C-terminal linker region and the chitin-binding domain, did not hydrolyze silkworm alpha-chitin. These results demonstrate that fish and insect chitinases possess unique substrate specificities that are correlated with their physiological roles in the digestion of food or cuticle.

Acetylation↗
PubMed2003

Isolation of BsoBI restriction endonuclease variants with altered substrate specificity.

BsoBI is a thermophilic restriction endonuclease that cleaves the degenerate DNA sequence C/PyCGPuG (where/=the cleavage site and Py=C or T, Pu=A or G). In the BsoBI-DNA co-crystal structure the D246 residue makes a water-mediated hydrogen bond to N6 of the degenerate base adenine and was proposed to make a complementary bond to O6 of the alternative guanine residue. To investigate the substrate specificity conferred by D246 and to potentially alter BsoBI specificity, the D246 residue was changed to the other 19 amino acids. Variants D246A, D246C, D246E, D246R, D246S, D246T, and D246Y were purified and their cleavage activity determined. Variants D246A, D246S, and D246T display 0.2% to 0.7% of the wild-type cleavage activity. However, the substrate specificity of the three variants is altered significantly. D246A, D246S, and D246T cleave CTCGAG sites poorly. In filter binding assays using oligonucleotides, wild-type BsoBI shows almost equal affinity for CTCGAG and CCCGGG sites. In contrast, the D246A variant shows 70-fold greater binding affinity for the CCCGGG substrate. Recycled mutagenesis was carried out on the D246A variant, and revertants with enhanced activity were isolated by their dark blue phenotype on a dinD Colon, two colons lacZ DNA damage indicator strain. Most of the amino acid substitutions present within the revertants were located outside the DNA-protein interface. This study demonstrates that endonuclease mutants with altered specificity and non-lethal activity can be evolved towards more active variants using a laboratory evolution strategy.

Amino Acid Substitution↗
PubMed1995

Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans.

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G2 to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G1 but high during S and G2 phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

Amino Acid Sequence↗
PubMed1997

Distinct substrate specificities of five human alpha-1,3-fucosyltransferases for in vivo synthesis of the sialyl Lewis x and Lewis x epitopes.

Five different human alpha-1,3-fucosyltransferase genes, the Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI and Fuc-TVII genes, have been cloned to date. We transfected HeLa cells and Namalwa cells with each of the five different genes, and established a series of stable cloned transformant cells. Thin-layer chromatography immunostaining analysis revealed that all five enzymes were able to synthesize sialyl Lewis x (sLe(x)) epitopes on glycolipids in HeLa cells, but each enzyme showed a different preference as to the carbohydrate chain length on glycolipids as acceptor substrates. Fuc-TIII and Fuc-TV showed very similar patterns of sLe(x) positive bands, which indicated that the enzymes have similar acceptor substrate specificities. Fuc-TVI exhibited a little different pattern from those of the former two enzymes. Fuc-TIV and Fuc-TVII showed similarity in the positive bands, however, their patterns were quite different from those of the former three enzymes. Four enzymes except for Fuc-TVII were able to synthesize the Lewis x (Le(x)) epitope on glycolipids in HeLa cells. Fuc-TV alone showed a little different pattern of Le(x) positive bands from those of the other three enzymes. Flow cytometric analysis of HeLa cells and Namalwa cells again demonstrated the similar specificities of Fuc-TIII and Fuc-TV. They exhibited similar stronger staining with FH6 (anti-sLe(x)) antibodies than that with the other enzymes. A phylogenetic tree of the five enzymes constructed using the neighbor-joining method showed good agreement with the similarities in the enzyme substrate specificity.

Burkitt Lymphoma↗
PubMed1998

Protein tyrosine kinases: structure, substrate specificity, and drug discovery.

Protein tyrosine kinases (PTKs) play a crucial role in many cell regulatory processes. It is therefore not surprising to see that functional perturbation of PTKs results in many diseases. Despite the diverse primary structure organization of various PTKs, the catalytic or kinase domains of various PTKs as well as that of Ser/Thr kinases are generally conserved. The high resolution crystal structure of a few PTKs has been solved in the last few years. In contrast to the well-defined linear peptide substrate motifs recognized by specific Ser/Thr kinases, the identification of specific substrate motifs for PTK has been slow. It is not until recently that through the use of combinatorial peptide library methods that specific recognition motifs for specific PTKs have begun to emerge. Efficient and specific peptide substrates for some PTKs with Km at the mid microM range have been identified. Based on these peptide substrates, relatively potent (IC50 at the low microM range) and highly selective pseudosubstrate-based peptide inhibitors have been developed. There has been enormous effort in the development of PTK inhibitors for diseases such as cancer, psoriasis, and osteoporosis. Several new high-throughput PTK assay technologies have recently been described. Small molecules against specific PTK have been developed. Most of them are competitive inhibitors at the ATP binding site. Some of these inhibitors have already been in clinical trial.

Amino Acid Sequence↗
PubMed1995

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R (GlcNAc to Man) beta 1,6- N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc beta 1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc beta 1-2Man alpha 1-6Glc/Man beta-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man alpha 1-3 residue and the 4-hydroxyl of the Man beta- residue of the Man alpha 1-6(Man alpha 1-3)Man beta-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAc beta 1-2(4,6-di-O-methyl-)Man alpha 1-6Glc beta-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Animals↗
PubMed1992

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acid coenzyme A transferase from rat liver mitochondria.

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acids coenzyme A transferase purified from rat liver mitochondria was assayed. In addition to the previously identified substrates succinyl-CoA, 3-hydroxy-3-methylglutaryl-CoA and malonyl-CoA (Francesconi et al.(1989) Biochim. Biophys. Acta, 999, 163-170) also methylmalonyl-CoA, glutaryl-CoA and adipyl-CoA acted as enzyme substrates, with the latter thioester showing the highest apparent affinity. All corresponding dicarboxylic acids, but not oxaloacetic, citric, alpha-ketoglutaric, malic, fumaric and glutamic acids, acted as coenzyme A acceptor substrates. None of the tested monocarboxylic acids, or the corresponding coenzyme A esters, were enzymatically transformed by the here described coenzyme A transferase.

Animals↗
PubMed2004

The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity.

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184-210 and 554-730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGDelta184-210 and XPGDelta554-730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.

Amino Acid Sequence↗
PubMed1997

Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of mu-calpain in human platelets.

The intracellular thiol protease mu-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit. Activation of mu-calpain requires calcium influx across the plasma membrane and the subsequent autoproteolytic conversion of the 80-kDa large subunit to a 78-kDa "intermediate" and a 76-kDa fully autolyzed form. Currently, there is limited information on the substrate specificities and functional roles of these distinct active forms of mu-calpain within the cell. Using antibodies that can distinguish among the 80-, 78-, and 76-kDa forms of mu-calpain, we have demonstrated a close correlation between the autolytic generation of the 78-kDa enzyme and the proteolysis of the non-receptor tyrosine phosphatase, PTP-1B, in ionophore A23187-stimulated platelets. Time course studies revealed that pp60(c-)src proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of mu-calpain (76 kDa). In vitro proteolysis experiments with purified mu-calpain and immunoprecipitated PTP-1B or pp60(c-)src confirmed selective proteolysis of pp60(c-)src by the 76-kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of mu-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of mu-calpain have demonstrated that the initial conversion of the mu-calpain large subunit to the 78-kDa form is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of mu-calpain (76 kDa) is responsible for the shedding of procoagulant-rich membrane vesicles from the cell surface. These studies demonstrate the existence of multiple active forms of mu-calpain within the cell, that have unique substrate specificities and distinct functional roles.

Antibodies↗
PubMed1991

Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity.

Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000. An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000. The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits. Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP. The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined. The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1). The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism.

Molecular Weight↗
PubMed2000

Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library.

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.

Blotting, Western↗
PubMed1997

X-ray structure of human class IV sigmasigma alcohol dehydrogenase. Structural basis for substrate specificity.

The structural determinants of substrate recognition in the human class IV, or sigmasigma, alcohol dehydrogenase (ADH) isoenzyme were examined through x-ray crystallography and site-directed mutagenesis. The crystal structure of sigmasigma ADH complexed with NAD+ and acetate was solved to 3-A resolution. The human beta1beta1 and sigmasigma ADH isoenzymes share 69% sequence identity and exhibit dramatically different kinetic properties. Differences in the amino acids at positions 57, 116, 141, 309, and 317 create a different topology within the sigmasigma substrate-binding pocket, relative to the beta1beta1 isoenzyme. The nicotinamide ring of the NAD(H) molecule, in the sigmasigma structure, appears to be twisted relative to its position in the beta1beta1 isoenzyme. In conjunction with movements of Thr-48 and Phe-93, this twist widens the substrate pocket in the vicinity of the catalytic zinc and may contribute to this isoenzyme's high Km for small substrates. The presence of Met-57, Met-141, and Phe-309 narrow the middle region of the sigmasigma substrate pocket and may explain the substantially decreased Km values with increased chain length of substrates in sigmasigma ADH. The kinetic properties of a mutant sigmasigma enzyme (sigma309L317A) suggest that widening the middle region of the substrate pocket increases Km by weakening the interactions between the enzyme and smaller substrates while not affecting the binding of longer alcohols, such as hexanol and retinol.

Alcohol Dehydrogenase↗
PubMed1990

Structural heterogeneity of sugar chains in immunoglobulin G. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases.

The heterogeneous asparagine-linked sugar chains of bovine and human immunoglobulins G were separated into 12 components by reversed-phase high performance liquid chromatography, and their structures were determined by 1H NMR spectroscopy. Both immunoglobulin (Ig) G sources contained eight non-bisected biantennary complexes and four bisected biantennary complexes. In the non-bisected sugar chains of bovine IgG, galactosylation of the Man alpha 1-3 branch predominated over that of the Man alpha 1-6, whereas in the bisected complexes galactosylation of the Man alpha 1-6 branch predominated. This difference can be explained by the substrate specificities of the galactosyl-transferases and of the N-acetylglucosaminyltransferase III involved in their synthesis. The sugar chains of human IgG1 differs in the distribution of its galactose residues from bovine IgG and human IgG2. The Man alpha 1-6 branch of all IgG1s was more highly galactosylated than the Man alpha 1-3 branch even in the non-bisected complexes. Such findings are in conflict with the substrate specificities of galactosyltransferases. Whereas these enzymes derivatized more of the Man alpha 1-6 branch of native human IgG1, in denatured protein more of the Man alpha 1-3 branch was galactosylated. Thus, protein conformation may influence the structure of its sugar chains.

Animals↗