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J Bubis

Publications and source records attributed to J Bubis.

At least 19 recordsLinked to original sources
PubMed2006

Field evaluation of four novel enzyme immunoassays for Chagas' disease in Venezuela blood banks: comparison of assays using fixed-epimastigotes, fixed-trypomastigotes or trypomastigote excreted-secreted antigens from two Trypanosoma cruzi strains.

Many serological tests have been developed for the diagnosis of Chagas' disease, but few have been subjected to a rigorous field evaluation. We have recently described several novel enzyme immunoassays (EIAs) based on fixed-whole organisms or trypomastigote excretory-secretory antigens (TESA) from different Trypanosoma cruzi strains (Tulahuen or Brazil). This study evaluated the most promising of these novel assays (e.g. fixed-epimastigotes, fixed-trypomastigotes, TESA Brazil and TESA Tulahuen antigens) in a field study of Venezuelan blood bank specimens. The assays were tested in an operator-blinded fashion using 2038 blood bank samples obtained from low and high T.cruzi prevalence regions of Venezuela (n= 1050 and n= 988 from Bolivar and Portuguesa states, respectively). Based on National Laboratory for Chagas Immunodiagnosis (NLCI) 'gold standard' results, all novel EIAs were superior to the commercial kit currently used in Venezuela, achieving 100% sensitivity and >99% specificity at optimal cut-off values. The novel assays identified seven false-negative samples compared with the routine screening performed by the Venezuelan blood bank although two samples were also misclassified as positive. Minor differences in the performance of the four novel assays were observed at lower arbitrary cut-off values. This study confirms the potential utility of both the fixed-organism and the TESA-based assays in the diagnosis of T.cruzi infection.

Adolescent↗
PubMed2005

Tight binding between a pool of the heterodimeric alpha/beta tubulin and a protein kinase CK2 in Trypanosoma cruzi epimastigotes.

Tubulin is the predominant phosphoprotein in Trypanosoma cruzi epimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-alpha tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 alpha-subunit antibodies immunoprecipitated radioactively labelled alpha and beta tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 alpha-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric alpha/beta tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that the T. cruzi tubulin-CK2 complex is globular with an estimated size of 145.4-147.5 kDa.

Animals↗
PubMed2002

Purification of a 64 kDa antigen from Trypanosoma evansi that exhibits cross-reactivity with Trypanosoma vivax.

Trypanosoma evansi and Trypanosoma vivax are the most extensively distributed trypanosomes responsible for diseases in livestock. Western blot and indirect immunofluorescence assays revealed a high immunological cross-reaction between these two parasites. An antigen with an apparent molecular mass of 64 kDa (p64), which exhibited cross-reactivity with T. vivax, was purified to homogeneity from a Venezuelan isolate of T. evansi. This antigen is glycosylated, contains a glycosyl-phosphatidylinositol anchor and appeared to be localized through the cell except in the nucleus, indicating that it could primarily be confined to the parasite surface. These results, together with its relative abundance and apparent molecular weight, suggest that p64 probably corresponds to the soluble form of a variable surface glycoprotein from T. evansi. Anti-p64 polyclonal antibodies, raised on mice, recognized a 53 kDa polypeptide band from a Venezuelan isolate of T. vivax on Western blots. Additionally, sera obtained from naturally infected animals also recognized p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, demonstrating that another relevant cross-reacting epitope, different than the inositol-1,2-cyclic phosphate of the cross-reacting determinant, must exist in p64. To date, p64 represents the first antigen isolated and partially characterized from T. evansi.

Animals↗
PubMed2001

Chemical modification of transducin with iodoacetic acid: transducin-alpha carboxymethylated at Cys(347) allows transducin binding to Light-activated rhodopsin but prevents its release in the presence of GTP.

Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [(3)H]IAA was incorporated per mole of T. Cys(347), located on the alpha-subunit of T (T(alpha)), was identified as the major labeled residue in the [(3)H]IAA-modified holoenzyme. In contrast, Cys(135) and Cys(347) were modified with [(3)H]IAA in the isolated T(alpha). IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233-242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be "locked" in the GDP-bound state of T, even in the presence of R*.

Animals↗
PubMed2001

Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin.

The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.

Animals↗
PubMed1998

Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Animals↗
PubMed1995

Improved purification of transducin subunits from bovine retinal rod outer segments.

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin.

Animals↗
PubMed1993

Identification of guanine nucleotide binding proteins from Trypanosoma cruzi.

Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2% Triton X-100 and 0.2% Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes.

Animals↗
PubMed1993

cAMP receptor protein from Trypanosoma cruzi: purification and cloning of a short sequence of the corresponding cDNA.

cAMP is involved in the differentiation of Trypanosoma cruzi, the causative agent of Chagas' disease. cAMP levels are elevated in the infective, non-dividing metacyclic trypomastigote stage, with respect to the non-infective, proliferative, epimastigote stage. In both stages three is a cAMP receptor protein (CARPT), with unique properties that differentiate it from the regulatory subunits of the cAMP-dependent protein kinase (RI and RII). The CARPT from T. cruzi epimastigotes was purified using ion-exchange chromatography, affinity chromatography and gel filtration. After the final step of purification, two protein bands were obtained, p89 and p70, corresponding to the intact CARPT and its proteolytic product. These two CARPT polypeptides were utilized to prepare polyclonal antibodies in rabbits. Previous results from our laboratory showed that CARPT cross-reacts with polyclonal antibodies prepared against the regulatory subunit (RII) of the cAMP-dependent protein kinase (PKA). As expected from these results, the anti-CARPT antibody recognized purified RII protein in an ELISA assay. The anti-CARPT antibodies were used for immunoblot analyses of epimastigote lysates. The two bands corresponding to the CARPT (p89 and p70), as well as a p40 band, were recognized. Immunoscreening of a T. cruzi lambda ZAP cDNA library with these anti-CARPT polyclonal antibodies yielded one positive clone (pBSCARPT) which contained a 540 bp insert. Northern analyses using the pBSCARPT clone as a probe, showed a 5.2 kb mRNA band in epimastigotes, which were grown in culture from 2 to 10 days in LIT medium. Sequence analyses of the 540 bp insert have failed to show homology to other gene sequences in the database.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Sites of interaction in the complex between beta- and gamma-subunits of transducin.

Transducin, the guanine nucleotide-binding regulatory protein in rod outer segments, is a heterotrimer consisting of alpha-, beta-, and gamma-subunits. Activation of the photoreceptor, rhodopsin, by light, results in activation of transducin which cleaves to form transducin alpha. GTP and a complex of beta gamma-subunits. We have investigated the point(s) of contact between the subunits of transducin by analyzing for the formation of intersubunit disulfide bond(s) in the presence of copper phenanthroline. The formation of a new species with an apparent molecular mass of 43 kDa was observed which had resulted from the formation of a disulfide bond between the beta- and gamma-subunits. The amino acid residues participating in the disulfide bond were identified as Cys-25 in the beta-subunit and Cys-36 and/or Cys-37 in the gamma-subunit. Thus, these cysteine residues and, probably, some of the adjacent amino acid residues form a point of contact between the beta- and gamma-subunits of transducin in the stable complex.

Amino Acid Sequence↗
PubMed1988

Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase.

The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.

Amino Acid Sequence↗
PubMed1988

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A.

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.

Affinity Labels↗
PubMed1988

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase.

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.

Adenosine Monophosphate↗
PubMed1988

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond.

The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.

Amino Acid Sequence↗
PubMed1988

Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I.

The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

CAMP-dependent protein kinase: prototype for a family of enzymes.

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.

Amino Acid Sequence↗
PubMed1987

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I.

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.

Amino Acid Sequence↗