Substrate-dependent dissociation of malate thiokinase.

Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure.     

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.     

Purification and characterization of a type II casein kinase from Drosophila melanogaster

A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution.     

Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains

Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subunit structure of Pseudomonas cytochrome oxidase.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.     

Structure and function of RNA replicase of bacteriophage Qbeta.

(1) The RNA replicase induced by bacteriophage Qbeta consists of four non-identical subunits designated as alpha (mol. wt. 74000), beta (mol. wt. 64000), gamma (mol. wt. 47000) and delta (mol. wt. 33000), only one (subunit beta) of which is specified by the phage genome. (2) Subunit alpha (30 S ribosomal protein "S1" as well as translational interference factor "i") is required only for (+) strand-directed RNA synthesis in the presence of the host factor. (3) Qbeta replicase lacking subunit alpha (R-alpha) is capable of replicating templates other than (+) strand, such as (--), "6S" RNA, poly(C) etc., in the absence of the host factor. (4) Subunit beta is suggested to be the nucleotide-polymerizing enzyme, but is unable to initiate RNA synthesis by itself. (5) Subunits gamma and delta are identical to the protein synthesis elongation factors, EF-Tu and EF-Ts, respectively, and are required only for initiation of RNA synthesis, but not for elongation. (6) A model of Qbeta replicase is presented in order to discuss observed template-enzyme interactions.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.     

Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.     

Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.     

Purification of two hexosaminidases from human kidney.

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.     

Limited proteolysis of the tryptophanyl-tRNA synthetase.

Earlier studies have shown that native tryptophanyl-tRNA synthetase from beef pancreas is composed of two apparently identical subunits having a molecular weight of 60000 plus or minus 2000 each. Incubation of the pruified enzyme with trypsin under restrictive conditions results in splitting of each subunit to form an enzymatically inactive polypeptide chain of mol. wt 24500 plus or minus 1500. During proteolysis, two distinct intermediate forms of mol. wt 51000 plus or minus 2000 and 40000 plus or minus 2000 and fragments of mol. wt 14000 plus or minus 2500 are formed. The presence of substrates, viz. ATP, tryptophan or tryptophanyl adenylate, decreases the rate of proteolysis. However, a band pattern monitored by acrylamide gel electrophoresis is qualitatively indistinguishable from that obtained in the absence of substrates. Native and trypsin-modified subunits (the latter having a molecular weight of 24500) have been maleylated, reduced, carbosymethylated and subjected to exhaustive digestion by trypsin followed by peptide mapping. Comparison of the finger prints has shown that the trypsin-modified subunit represents a polypeptide with lowered content of dicarboxylic amino acids. That the number of peptides revealed after complete proteolysis of native and trypsin-modified subunits does not favour the presence of long repetitive sequences in each subunit, is at variance with some bacterial aminoacyl-tRNA synthetases. Study of the fluorescence polarisation of 1-anilino-8-napthalene sulphonate adsorbed on the dimeric tryptophanyl-tRNA synthetase, indicates that the molecule behaves as a complete entity in Brownian rotation. The trypsin-resistant end products, composed of two types of polypeptides (mol. wts 24500 and 14000), remain associated with each other. From the mol. wt of this associate it follows that each fragment is present in the associate in duplicate. When the purification procedure was carried out in the absence of a protease inhibitor, the active modified enzyme form was obtained. As judged from the molecular weight values, it is composed of two equal subunits corresponding to one of the products of limited proteolysis. The data presented are compatible with compact three-dimensional structure of tryptophanyl-tRNA synthetase having very limited regions exposed to exogenous or endogenous proteolysis.     

The role of dissimilar subunits of NAD-specific isocitrate dehydrogenase from pig heart. Evaluation using affinity labeling

NAD-specific isocitrate dehydrogenase from pig heart is composed of three dissimilar subunits present in the native enzyme as 2 alpha:1 beta: 1 gamma, with a tetramer being the smallest form of complete enzyme. The role of these subunits has been explored using affinity labeling. Specifically labeled subunits are separated and then recombined with unmodified subunits to form dimers. Recombination of beta or gamma subunits modified by the isocitrate analogues, 3-bromo-2-ketoglutarate and 3,4-didehydro-2-ketoglutarate, with unmodified alpha subunit led to the same activity in the dimer as when unmodified beta or gamma was combined with alpha. Contrastingly, modification of alpha with these isocitrate analogues led to loss in activity either alone or when recombined with beta or gamma. Hence, the isocitrate site on alpha is required for catalytic activity but the isocitrate sites on beta or gamma are not necessary for the activity of the functional dimer. Reaction of isolated subunits with 3-bromo-2-ketoglutarate shows that alpha and the alpha beta dimer are modified at about the same rate as holoenzyme, suggestive of similarity of the isocitrate site in native enzyme and in isolated active entities containing alpha subunit; in contrast, beta and gamma subunits react more slowly. Modification by the 2',3'-dialdehyde derivative of the allosteric effector, ADP, led to loss of activity in reconstituted dimers, independent of which subunit was modified. Reaction of isolated subunits with the dialdehyde derivative of ADP is slow compared to the initial reaction with native enzyme, indicating differences in the effects of ADP on intact enzyme and subunits. The ADP sites on all subunits may thus be important in intersubunit interactions, which in turn modulate catalytic activity.     

Assembly of Torpedo acetylcholine receptors in Xenopus oocytes

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.     

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.     

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts.

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.