Gln Residues Research Articles

Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death.

Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase.

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

K+-independent Gastric H+,K+-ATPase Activity: DISSOCIATION OF K+-INDEPENDENT DEPHOSPHORYLATION AND PREFERENCE FOR THE E1 CONFORMATION BY COMBINED MUTAGENESIS OF TRANSMEMBRANE GLUTAMATE RESIDUES

Several mutations of residues Glu(795) and Glu(820) present in M5 and M6 of the catalytic subunit of gastric H(+),K(+)-ATPase have resulted in a K(+)-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E(1) conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu(343), present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K(+)-activated ATPase activity and an E(2) preference and a Gln giving K(+)-independent ATPase activity and an E(1) preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E(2) <--> E(1) equilibrium was still dominated by the 820 residue: a Glu giving E(2) and a Gln giving E(1). This indicates that the preference for the E(1) conformation of the E820Q mutation is independent of an active dephosphorylation process.

Bovine pancreatic elastase II cleaves Gln-Ile bond.

A peptidase (GICP) that cleaves the Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a sequence in phenylalanine oxidase, was purified from bovine pancreas. The purified enzyme had an Mr of approximately 29,000, as determined by SDS-PAGE, and its N-terminal sequence was identical to that of bovine pancreatic elastase II. The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1 (Km = 8.3 microM k(cat) = 2.1 s(-1)) and the catalytic efficiency (2.6 X 10(5) M(-1)s(-1)) was comparable to those of elastase II from porcine pancreas and rat mesenteric arterial bed perfusate. The P1 site specificity of GICP toward oxidized insulin A and B chains suggested that major cleavage sites were the peptide bond at the C-terminal side of Gln, Leu, His, and Tyr residues.

Identification of Residues of the Saccharomyces cerevisiae G Protein-coupled Receptor Contributing to α-Factor Pheromone Binding

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.

Gas phase conformation can have an influence on peptide fragmentation.

In the post-genomic era, mass spectrometry is destined to fulfil a central role in biomedical research, and it is in the area of protein identification that mass spectrometry is now most rapidly expanding. An important identification method is to subject a protein to proteolysis and determine the resulting peptide masses and/or primary structure. From such determinations proteins can be identified. Tandem mass spectrometry (MS/MS) is used to determine primary structure and, for high-throughput identification, computer-based automated strategies are a prerequisite. Computer programs are available for such identifications, where simulated MS/MS spectra of amino acid sequences within a database are generated and compared to experimental spectra. Such algorithms take into account empirical rules for peptide fragmentation, rather than specific gas-phase ion chemistry. For example, fragmentation of each peptide bond is usually considered to be equally facile. In reality, this is not the case. Gas-phase ion chemistry bears an important role in determining the abundance of fragment ions in MS/MS spectra. In this communication, the gas-phase ion chemistry responsible for the facile cleavage between Gln and Gly residues is investigated, particularly in relation to Proline Rich Protein-1.

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 Å resolution. Because of the most alkaliphilic nature and it’s highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

Synthesis and conformational studies of a 1,1′-ferrocenophane lactam mimetic of substance P

The synthesis of a bis-phenylalanine mimetic 6 and its incorporation into Substance P (SP), giving a conformationally constrained organometallic SP analogue 8, is described. The lactam 6 was synthesized in five steps, via a HWE olefination reaction, enantioselective hydrogenation with [Rh(I)(COD)(( S, S)Et-DuPHOS)] +OTf − and intramolecular cyclization with PyAOP as a coupling reagent. Comparative CD studies of 8 with native SP indicated that the flexibility around the amide bond of Phe(7)–Phe(8) sequence is crucial for the C-terminal (from residue Gln(4)) to adopt an α-helical conformation in the micellar environment created by SDS or in methanol.

Role of medium- and long-range interactions in discriminating globular and membrane proteins

The analysis of inter-residue interactions in protein structures provides considerable insight to understand their folding and stability. We have previously analyzed the role of medium- and long-range interactions in the folding of globular proteins. In this work, we study the distinct role of such interactions in the three-dimensional structures of membrane proteins. We observed a higher number of long-range contacts in the termini of transmembrane helical (TMH) segments, implying their role in the stabilization of helix–helix interactions. The transmembrane strand (TMS) proteins are having appreciably higher long-range contacts than that in all-β class of globular proteins, indicating closer packing of the strands in TMS proteins. The residues in membrane spanning segments of TMH proteins have 1.3 times higher medium-range contacts than long-range contacts whereas that of TMS proteins have 14 times higher long-range contacts than medium-range contacts. Residue-wise analysis indicates that in TMH proteins, the residues Cys, Glu, Gly, Pro, Gln, Ser and Tyr have higher long-range contacts than medium-range contacts in contrast with all-α class of globular proteins. The charged residue pairs have higher medium-range contacts in all-α proteins, whereas hydrophobic residue pairs are dominant in TMH proteins. The information on the preference of residue pairs to form medium-range contacts has been successfully used to discriminate the TMH proteins from all-α proteins. The statistical significance of the results obtained from the present study has been verified using randomized structures of TMH and TMS protein templates.

Transglutaminase-mediated cross-linking of α-crystallin: structural and functional consequences

Aggregation and covalent cross-linking of the crystallins, the major structural proteins of the eye lens, increase light scattering by the lens leading to opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis. Calcium-activated transglutaminase (TG)-catalyzed cross-linking of some lens proteins has been reported earlier. We show here that α-crystallin, a major structural protein in the lens and a member of the small heat shock protein family, is also a substrate for TG-mediated cross-linking, indicating the presence of donor Lys and acceptor Gln residues in the protein. Upon TG-catalyzed dimerization, the secondary and tertiary structures of the protein are altered, and its surface hydrophobicity reduced. The chaperone-like property of the protein, suspected to be one of its functions in situ, is substantially reduced upon such cross-linking. These results, taken together with earlier ones on lens β-crystallins and vimentin, suggest that TG-mediated events might compromise lens function. Also, since α-crystallin occurs not only in the lens but in other tissues as well, such TG-catalyzed cross-linking and the associated alterations in its structure and activity would be of general pathological interest.

Crosslinking of alpha 2-antiplasmin to fibrin.

Human alpha 2-antiplasmin (alpha 2AP) is the primary inhibitor of plasmin-mediated fibrinolysis and is an efficient substrate of activated factor XIII (FXIIIa). Among 452 amino acid residues in alpha 2AP, Gln2 is believed to be the sole FXIIIa-reactive site that participates in crosslinking alpha 2AP to fibrin. We studied the effect of mutating Gln2 on the ability of FXIIIa to catalyze crosslinking of alpha 2AP to fibrin. By FXIIIa catalysis, [14C]methylamine was incorporated into a Q2A-alpha 2AP mutant in which Gln2 (Q) was replaced by Ala (A), thereby indicating that wildtype alpha 2AP has more than one FXIIIa-reactive site. To identify the FXIIIa-reactive sites in alpha 2AP, wildtype alpha 2AP and Q2A-alpha 2AP were labeled with 5-(biotinamido)pentylamine by FXIIIa. Each labeled alpha 2AP was digested with trypsin and applied to an avidin affinity column to capture labeled peptides. Edman sequencing and mass analysis of each labeled peptide showed that out of 35 Gln residues in wildtype alpha 2AP, four were labeled with the following order of efficiency: Gln2 > Gln21 > Gln419 > Gln447. Q2A-alpha 2AP was also labeled at the three minor sites, Gln21 > Gln419 > Gln447. Q2A-alpha 2AP became crosslinked to fibirin(ogen) by FXIIIa catalysis at approximately one-tenth the rate of wt-alpha 2AP. These results demonstrate that alpha 2AP has one primary (Gln2) and three minor substrate sites for FXIIIa and that the three minor sites identified in this study can also participate in crosslink formation between alpha 2AP and fibrin, but at a much lower efficiency than the Gln2 site.

Buried polar residues in coiled-coil interfaces.

Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices. Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged. We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position. The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined. The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes.

Gas-Phase Conformation Can Have an Influence on Peptide Fragmentation

In the post-genomic era, mass spectrometry is destined to fulfil a central role in biomedical research, and it is in the area of protein identification that mass spectrometry is now most rapidly expanding. An important identification method is to subject a protein to proteolysis and determine the resulting peptide masses and/or primary structure. From such determinations proteins can be identified. Tandem mass spectrometry (MS/MS) is used to determine primary structure and, for high-throughput identification, computer-based automated strategies are a prerequisite. Computer programs are available for such identifications, where simulated MS/MS spectra of amino acid sequences within a database are generated and compared to experimental spectra. Such algorithms take into account empirical rules for peptide fragmentation, rather than specific gas-phase ion chemistry. For example, fragmentation of each peptide bond is usually considered to be equally facile. In reality, this is not the case. Gas-phase ion chemistry bears an important role in determining the abundance of fragment ions in MS/MS spectra. In this communication, the gas-phase ion chemistry responsible for the facile cleavage between Gln and Gly residues is investigated, particularly in relation to Proline Rich Protein-1.

Analysis of a data set of paired uncomplexed protein structures: New metrics for side‐chain flexibility and model evaluation

We compiled and analyzed a data set of paired protein structures containing proteins for which multiple high-quality uncomplexed atomic structures were available in the Protein Data Bank. Side-chain flexibility was quantified, yielding a set of residue- and environment-specific confidence levels describing the range of motion around chi1 and chi2 angles. As expected, buried residues were inflexible, adopting similar conformations in different crystal structure analyses. Ile, Thr, Asn, Asp, and the large aromatics also showed limited flexibility when exposed on the protein surface, whereas exposed Ser, Lys, Arg, Met, Gln, and Glu residues were very flexible. This information is different from and complementary to the information available from rotamer surveys. The confidence levels are useful for assessing the significance of observed side-chain motion and estimating the extent of side-chain motion in protein structure prediction. We compare the performance of a simple 40 degrees threshold with these quantitative confidence levels in a critical evaluation of side-chain prediction with the program SCWRL.

Oligomerization of Rac1 GTPase Mediated by the Carboxyl-terminal Polybasic Domain

The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.

A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.

Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.

Identification of insulin domains important for binding to and degradation by endosomal acidic insulinase.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([Asp(B10)]- and [Glu(A13),Glu(B10)]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC(50) values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (Glu(B13)-Leu(B17)) and at the C-terminus (Arg(B22)-Thr(B27)), the latter region comprising the initial cleavages at Phe(B24)-Phe(B25) (major pathway) and Phe(B25)-Tyr(B26) (minor pathway) bonds. Except for the [Glu(A13),Glu(B10)]-insulin mutant, only one cleavage on the A-chain was observed at residues Gln(A15)-Leu(A16). Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B(22)-B(30) peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have identified a highly specific molecular interaction of insulin with EAI at the aromatic locus Phe(B24)-Phe(B25)-Tyr(B26). Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]Tyr(A14)-H2-analog, a protease-resistant insulin analog, undergoes a greater lysosomal transfer and lesser degradation than [(125)I]Tyr(A14)-insulin, confirming that endosomal sorting is regulated directly or indirectly by endosomal proteolysis.

A Specific Isozyme of 2′-5′ Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5' linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx(L) in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx(L) interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

Comparison of Deamidation Activity of Transglutaminases

ABSTRACT: A comparison was made of the deamidation activity of transglutaminases fro m guinea pig liver (GTGase), fish red sea bream liver (FTGase) and microorganisms (MTGase). Against the Z‐Gln‐Gly, kinetic constants of the deamidation and incorporation of primary amine were measured. GTGase and FTGase showed similar deamidation activity, however, that of MTGase was less than 1/7 of the other two TGases. Against the proteins, N, N‐dimethylated casein and native gliadin, FTGase was the most active and deamidated respectively 45.5% and 38.2% of Gln residues. The deamidation rate of these proteins by GTGase was less than 1/2 and these results were expected to be caused by the difference of substrate specificity of the TGases.