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Results for “Catalytic Domain”

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At least 19 recordsLinked to original sources

PubMed1992

Expression of the phosphorylase kinase gamma subunit catalytic domain in Escherichia coli.

The catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli. The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain. Full-length protein was produced in insoluble aggregates. Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component. Full-length protein could not be refolded successfully. The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies. The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol. wt 32,000 +/- 2000 with a high sp. act. of 5.3 mumol 32P incorporated into phosphorylase b(PPB)/min/nmol. Kinetic parameters gave Km for ATP 46 +/- 3 microM and Km for PPb 27 +/- 1 microM. The sp. act. and the Km values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties. The ratio of activities at pH 6.8/8.2 was 0.84. gamma-trc was inhibited by ADP with a Ki of 52 microM and was sensitive to activation by Mg2+ and inhibition by Mn2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit. Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1992

Kinetic analysis of protein kinase C inhibition by staurosporine: evidence that inhibition entails inhibitor binding at a conserved region of the catalytic domain but not competition with substrates.

The indole carbazole staurosporine is an extraordinarily potent antiproliferative agent that inhibits the growth of cultured mammalian cells at concentrations of less than 1 nM. The antiproliferative activity of staurosporine is attributed to its potent inhibition of diverse protein kinases, but the mechanism of staurosporine inhibition has not been elucidated for any protein kinase. Protein kinase C (PKC) is a family of Ca(2+)- and phosphatidylserine-dependent protein kinases that are activated in vivo by the second messenger diacylglycerol. A fully active, Ca(2+)- and phosphatidylserine-independent, catalytic fragment of PKC that contains only the catalytic domain of the enzyme can be produced by limited proteolysis. Previous studies indicated that staurosporine inhibits PKC by binding its catalytic domain. In this study, we define the kinetics of inhibition by staurosporine of a catalytic fragment of rat brain PKC-gamma and of a catalytic fragment generated from a rat brain PKC-alpha/PKC-beta mixture. Our kinetic results provide evidence that staurosporine inhibits PKC by binding to a site of the catalytic domain other than the ATP substrate and protein substrate binding sites. Staurosporine inhibition appears to entail binding at a conserved site in the catalytic domain of PKC, because staurosporine inhibited rat brain PKC-alpha, PKC-beta, and PKC-gamma, as well as the catalytic fragments of PKC-beta and PKC-gamma, with similar protencies. The kinetics of inhibition of the catalytic fragment of PKC-gamma were uncompetitive with respect to histone III-S, providing evidence that the binding of histone III-S at the active site of the catalytic fragment precedes the binding of staurosporine to the enzyme. Taken in the context of previous mechanistic studies of PKC-catalyzed histone III-S phosphorylation, these results provide evidence that staurosporine binds to a complex of PKC, MgATP, and histone III-S, thereby forming a complex that cannot break down to products. In addition, the inhibitory kinetics observed when the ATP concentration was varied provided evidence that staurosporine reduces the affinity of MgATP for the catalytic fragment of PKC-gamma. Thus, the kinetics of inhibition of the catalytic fragment of PKC-gamma by staurosporine provide evidence that staurosporine inhibits PKC by a mixed mechanism.

Adenosine Triphosphate↗

PubMed2023

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

src-Family Kinases↗

PubMed1992

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

Development and characterization of a panel of monoclonal antibodies against the catalytic domain of the human fes proto-oncogene product.

In developing monoclonal antibodies (Moabs) against the human fes proto-oncogene product, recombinant DNA technology was used to target reactivity of the Moabs towards the catalytic domain of it. Therefore, sequences of human fes exons 15-19 encoding amino acid residues 612 to 822 which harbor the catalytic domain except the presumed ATP-binding region, were fused in phase to the bacterial trp E gene which encodes anthranilate synthase. After partial purification of it, the bacterially produced hybrid product of this trp E-delta fes fusion gene was used as immunogen. A series of twelve mouse Moabs was obtained which recognized the human p92fes protein and the viral oncogene product p85gag-fes encoded by the Snyder-Theilen strain of feline sarcoma virus. Reactivity appeared to be directed towards the catalytic domain of the human fes proto-oncogene product. This was demonstrated by in vitro transcription and translation experiments using human fes coding sequences from exons 16-19. Upon testing their functional activity in divers immunological techniques, the whole panel of Moabs appeared to be useful in immunoprecipitation, Western blot and immunohistochemical analysis. Immunocytochemical analysis indicated that p85gag-fes is predominantly a cytoplasmic protein.

Adenosine Triphosphate↗

PubMed1992

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

Amino Acid Sequence↗

PubMed1992

Guanine-nucleotide binding activity, interaction with GTPase-activating protein and solution conformation of the human c-Ha-Ras protein catalytic domain are retained upon deletion of C-terminal 18 amino acid residues.

A truncated human c-Ha-Ras protein that lacks the C-terminal 18 amino acid residues and the truncated Ras protein with the amino acid substitution Gly-->Val in position 12 were prepared by an E. coli overexpression system. The truncated Ras protein showed the same guanine-nucleotide binding activity and GTPase activity as those of the full-length Ras protein. Further, the same extent of GTPase activity enhancement due to GTPase-activating protein was observed for the truncated and full-length Ras proteins. In fact, two-dimensional proton NMR analyses indicated that the tertiary structure of the truncated Ras protein (GDP-bound or GMPPNP-bound) was nearly the same as that of the corresponding catalytic domain of the full-length Ras protein. Moreover, a conformational change around the effector region upon GDP-->GMPPNP exchange occurred in the same manner for both proteins. These observations indicate that the C-terminal flanking region (18 amino acid residues) of the Ras protein does not appreciably interact with the catalytic domain. Therefore, the truncated Ras protein is suitable for studying the molecular mechanism involved in the GTPase activity and the interaction with the GTPase-activating protein. On the other hand, an active form of the truncated Ras protein, unlike that of the full-length Ras protein, did not induce neurite outgrowth of rat pheochromocytoma PC12 cells. Thus, membrane anchoring of the Ras protein through its C-terminal four residues is not required for the interaction of Ras and GAP, but may be essential for the following binding of the Ras-GAP complex with the putative downstream target.

Animals↗

PubMed1992

Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli.

Human stromelysin is a member of the matrix metalloproteinase family involved in connective tissue degradation. The stromelysin catalytic domain (SCD) lacking both propeptide and C-terminal fragment was expressed in Escherichia coli in soluble and insoluble forms. The insoluble SCD was refolded to the active form in high yield. The protein showed remarkable thermal stability and was able to cleave a thiopeptolide substrate and its natural substrate proteoglycan. The stable and active 20-kDa protein provides an opportunity to elucidate the structure as well as the mechanism of catalysis and inhibition for matrix metalloproteinases.

Amino Acid Sequence↗

PubMed1992

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the bacterium Cellulomonas fimi.

Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees.

Actinomycetales↗

PubMed1992

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin.

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Amino Acid Sequence↗

PubMed1992

A mutation in the catalytic domain of pp60v-src is responsible for the host- and temperature-dependent phenotype of the Rous sarcoma virus mutant tsLA33-1.

We have analyzed a host- and temperature-dependent mutant of Rous sarcoma virus in order to learn more about the nature of mutations which lead to a host range phenotype. We have cloned and sequenced the v-src genes from this mutant, tsLA33-1, and from its presumed parent, tsLA33. Both the tsLA33 and the tsLA33-1 pp60v-src proteins contain multiple mutations. The tsLA33 v-src gene product has amino acid alterations at four positions. In the tsLA33-1 v-src gene product, two of these four mutations have reverted to wild type. We have constructed chimeras between the two mutant v-src gene products and between each mutant and the Prague A v-src gene product. To assess the contribution of each amino acid change to the transformation phenotypes of tsLA33 and tsLA33-1, we expressed the hybrid proteins in both chicken embryo fibroblasts and Rat-3 fibroblasts. Additionally, we have measured the protein tyrosine kinase activity of chimeras constructed between the tsLA33 and tsLA33-1 pp60v-src proteins. Our results indicate that mutations in the catalytic domain of each protein are the principal determinants of the transforming ability and protein tyrosine kinase activity of the tsLA33 and tsLA33-1 pp60v-src proteins.

Animals↗

PubMed1992

A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities.

A plant polysaccharide hydrolase cDNA, designated celD, was isolated from a cDNA library of the rumen fungus Neocallimastix patriciarum. The enzyme encoded by celD had endoglucanase, cellobiohydrolase and xylanase activities. Deletion analysis revealed that celD cDNA can be truncated to code for three catalytically active domains. Each domain had the same substrate specificity as the enzyme produced by the untruncated celD and also possessed cellulose-binding capacity. Substrate competition studies showed that carboxymethylcellulose and xylan appear to compete with methylumbelliferyl cellobioside for the same active site within each domain. Expression of celD transcript in the rumen fungus was constitutive and was not affected by the presence of cellulose in the culture medium.

Animals↗

PubMed1992

Purification and characterization of the catalytic domains of the human receptor-linked protein tyrosine phosphatases HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR).

Human HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR) are transmembrane receptor-like proteins whose cytoplasmic regions contain either one (HPTP beta) or two (LCA and LAR) domains that are homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain 1 has enzymatic activity, the membrane-distal domain 2 of both LCA and LAR has no detectable catalytic activity. The cytoplasmic regions of HPTP beta, LCA, and LAR were expressed in Escherichia coli and purified to greater than 90% purity. Modulatory effects of various low molecular weight compounds and homo- and copolymers of amino acids were examined. Several polypeptides that contain a high proportion of tyrosine were strongly inhibitory to these PTPases. To determine a possible role for the LAR domain 2, the properties of recombinant LAR PTPases containing both domains 1 and 2 (LAR-D1D2) or only domain 1 (LAR-D1) were compared. In nearly all aspects examined, LAR-D1 and LAR-D1D2 were indistinguishable. However, polycationic polypeptides strongly stimulated the PTPase activity of LAR-D1D2, but not LAR-D1, using the peptide substrate Raytide. Thus, basic polypeptides seem to indirectly alter the catalytic activity of domain 1 by interacting with domain 2. This result suggests that domain 2 has a regulatory function.

Amino Acid Sequence↗

PubMed1992

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.

Amino Acid Sequence↗

PubMed1992

Molecular structure of the multifunctional fatty acid synthetase gene of Brevibacterium ammoniagenes: its sequence of catalytic domains is formally consistent with a head-to-tail fusion of the two yeast genes FAS1 and FAS2.

The Brevibacterium ammoniagenes fatty acid synthetase (FAS) gene was isolated from a series of overlapping clones by both immunological and plaque hybridization screening of two independent gene libraries. From the isolated DNA a contiguous segment of 10,549 bp was sequenced in both directions. The sequenced DNA contained a very long (9312 nucleotides) open reading frame coding for a protein of 3104 amino acids and with a molecular mass of 327,466 daltons. Based on characteristic sequence motifs known from other FAS systems, seven different FAS active centres were identified at distinct locations within the polypeptide chain. Only one component enzyme, the 3-hydroxydecanoyl beta, gamma-dehydratase, has not yet been localized definitively within the gene. Translation is presumed to start from a GUG triplet located 25 nucleotides downstream of the transcriptional initiation site. There is a canonical Shine-Dalgarno sequence just before this start codon. Comparison of the B. ammoniagenes FAS sequence with those of other known fatty acid synthetases revealed a particularly high degree of similarity to the products of the two yeast genes, FAS1 and FAS2 (30% identical and 46% identical plus closely related amino acids). This similarity extends over the entire length of the genes and involves not only the primary sequences of individual component enzymes but also their sequential order within the multifunctional proteins. These data, together with those on the structure of other fatty acid synthetases, are interpreted in terms of a contribution of both primary structure and subunit cooperation to a conserved topology of functional domains common to all type I FAS complexes.

Amino Acid Sequence↗

PubMed1992

Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits.

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex). An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein. The extent of lipoylation was related to the degree of aeration during amplification. The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex. This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p. The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex. Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p. A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made. Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E. coli E2p is essential, whereas R603 could be replaced without inactivating E2p. Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain. The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.

Acetyltransferases↗

PubMed1992

Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain.

Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

O-glycosylation in Aspergillus glucoamylase. Conformation and role in binding.

Functional peptides have been produced by proteolysis of glucoamylase (glucan 1,4-alpha-glucosidase; EC 3.2.1.3) from Aspergillus niger and purified by affinity chromatography, gel filtration and two ion-exchange-chromatography steps. The peptides correspond to residues 499-616 and 509-616 of the original glucoamylase molecule. Together with G1C (residues 471-616 from glucoamylase 1) [Belshaw & Williamson (1990) FEBS Lett. 269, 350-353], the three peptides all contain the C-terminal domain (residues 509-616) but, in addition, contain different proportions of the O-glycosylated region. The properties of these peptides have been compared to define the function of the O-linked oligosaccharides in this protein. The O-glycosylated region plays only a minor role in binding to hydrogen-bond ordered starch. The difference between the apparent free energy (delta G) for binding between the non-glycosylated C-terminal domain (-26.0 kJ/mol) and the C-terminal domain containing the fully O-glycosylated region (-25.0 kJ/mol) is only 1.0 kJ/mol. Binding to beta-cyclodextrin suggests that even this difference may reflect a small conformational change in the C-terminal domain rather than a direct effect of the O-linked sugars. The c.d. spectrum of the O-glycosylated region is deduced by comparison of the three peptides and is predominantly that of a random-coil structure. Two-dimensional n.m.r. spectra of glucoamylase and of the glycosylated peptide 499-616 show that the binding domain is more mobile than the catalytic domain and that its mobility is further increased on removal of the catalytic domain. The O-glycosylated region is more mobile still, and there is a marked increase in its mobility on removal of the catalytic domain. The O-glycosylated region in the intact protein can therefore be envisaged as a semi-rigid rod. The results show that a major function of O-glycosylation in glucoamylase 1 is to provide an extended peptide backbone and hence a fixed distance in linking the catalytic and binding domains. It does not in itself significantly increase the binding affinity for starch.

Amino Acid Sequence↗