Propionicacidemia: absence of alpha-chain mRNA in fibroblasts from patients of the pccA complementation group.

Propionicacidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl-CoA carboxylase (PCC) activity. The enzyme has the structure alpha 4 beta 4, with the alpha chain containing a covalently bound biotin prosthetic group. Patients have been placed into two major complementation groups, pccA and pccBC, that may correspond to the genes encoding the alpha and beta chains of PCC. The pccBC group is further divided into two subgroups, pccB and pccC, apparently owing to intragenic complementation. We previously reported combined alpha- and beta-chain deficiency in pccA mutants and absence of beta chain in pccC and pccBC mutants after isotope-tracer labeling and immunoprecipitation of cultured-fibroblast extracts. Using cDNA clones coding for the alpha and beta chains as probes, we found absence of alpha mRNA in four of six pccA strains and presence of beta mRNA in all pccA mutants studied. We also found presence of both alpha and beta mRNAs in three pccBC, two pccB, and three pccC mutants. From these data, we confirm the gene assignments of the complementation groups (PCCA gene = pccA complementation group; PCCB gene = pccBC and subgroups) and support the view that pccA patients synthesize a normal beta chain that is rapidly degraded in the absence of complexing with alpha chains.     

Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations participating in interallelic complementation in propionic acidemia.

Deficiency of propionyl-CoA carboxylase (PCC; alpha 4 beta 4) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA (alpha-subunit) and PCCB (beta-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, we have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound heterozygote of Pro228Leu and Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS + 1 G-->T, located after Lys466. We suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, we previously had proposed that delta Ile408 was the complementing allele. We now show that its second allele, "Ins.Del," a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. We conclude that the interallelic complementation results from mutations in domains that can interact between beta-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, we suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site.     

Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli.

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.     

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.     

Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X-100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate-binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X-100 and purified by affinity chromatography on monomeric avidin-Sepharose. The enzyme is composed of three types of polypeptides: the group of alpha chains (Mr 120000-140000) containing the biotin, the beta chain (60000) and an apparently hydrophobic gamma chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The beta and gamma chains but not the alpha chain could be labelled by N-ethyl-[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000-20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl-CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.     

Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity. A genetic analysis was performed on 29 mutant strains harboring defects in the structural gene pyrB, encoding the catalytic chains of aspartate transcarbamoylase of Escherichia coli. Extracts from 15 strains contained intact, inactive enzyme-like molecules of the same size as the purified wild type enzyme. These same extracts contained a predominant polypeptide chain which migrated electrophoretically at the same rate as catalytic chains from wild type enzyme. In addition to these 15 different missense mutants, 14 others (presumably chain-terminating mutants) were isolated; no polypeptides corresponding to full length catalytic chains were detected in these strains. Based on their reversion and suppression properties, seven were designated as frameshift and two as amber nonsense. A fine structure recombination map of the pyrB locus was constructed from a series of three-factor transductional crosses. Mutational sites were correlated with regions in the polypeptide sequence by relating their map positions to that of mutation pyrB231 which results in an amino acid replacement at position 128. Moreover, since recent crystallographic studies indicate that residue 128 is located near the junction between the NH2- and COOH-terminal folding domains, the mutational sites can be placed within either of these two regions of tertiary structure. Interallelic complementation experiments showed four units of complementation. Those defining the alpha and beta units were missense mutants with their mutational sites in the NH2- and COOH-terminal domains, respectively. The mutants determining the delta and gamma units involved premature polypeptide chain termination and their mutational sites were correlated with distal regions of the two respective domains. Several mutants of the chain-terminating type failed to complement members of more than one unit. Possible effects of the various mutations and their implications for mechanisms of complementation and enzyme activity are presented.     

Properties of Bacteriophage T4 ribonucleoside diphosphate reductase subunits coded by nrdA and nrdB mutants

As a part of the study of the bacteriophage T4-induced deoxyribonucleotide synthetase complex, an investigation has been made of the T4 ribonucleoside diphosphate reductases formed by a series of mutants of nrdA and B, the genes coding, respectively, for the alpha 2 and beta 2 subunits of the enzyme. dATP affinity columns were used to isolate the enzyme by a single-step procedure. The molecular weights of the alpha and beta chains have been found to be 84,000 and 43,500, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since alpha 2 beta 2 is bound to dATP affinity columns through allosteric effector sites on alpha 2, it is possible to monitor the binding of beta 2 to alpha 2. dTTP- and ATP-Sepharose columns did not bind T4 alpha 2 beta 2, although the corresponding nucleoside triphosphates are effectors of the enzyme and although the alpha 2 subunit of the host enzyme binds to these columns. Missense mutants of nrdA and B forming alpha 2 and beta 2 subunits that lacked catalytic activity but retained the ability to form the alpha 2 beta 2 complex have been described. The 50,000-dalton fragment formed by an amber mutant of nrdA did not bind to the dATP affinity column, providing evidence that a region of the carboxyl-terminal segment of the alpha chain is required for retention. The beta 2 subunit appears to protect the alpha 2 protein. On infection by nrdB mutants not forming beta 2, the alpha protein chain was cleaved specifically to form 3 protein chains of 61,000, 57,000, and 24,500 daltons, which retain the ability to bind to dATP-Sepharose. Some effects of mutation on the interaction of the alpha and beta chains of the enzyme with the deoxyribonucleotide synthetase complex have been examined.     

Tet protein domains interact productively to mediate tetracycline resistance when present on separate polypeptides

Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.     

A previously unrecognized subunit of the receptor for immunoglobulin E

Our laboratory previously found that under conditions that stabilized the interaction between the alpha and beta subunits of the receptor for immunoglobulin E, two new components were recovered having apparent molecular weights of 45 000 and 20 000, respectively. In this paper, we characterize the 20-kDa material. We demonstrate that it consists of a disulfide-linked dimer of 10-kDa polypeptides and that these have all the characteristics expected for subunits of the receptor. We propose that they be termed gamma chains and that the receptor consists of four chains: one alpha, one beta, and two gamma chains. The gamma chains share many of the labeling properties of the beta chain and, like the latter, are likely to be embedded in the plasma membrane and exposed on the internal but not the external surface of the bilayer.     

Immunological comparison of purified DNA polymerase alpha from embryos of Drosophila melanogaster with forms of the enzyme present in vivo

Specific antisera to purified DNA polymerase alpha from embryos of Drosophila melanogaster and to two of the four constituent subunits (alpha, beta, gamma, and delta) were prepared. These antibodies have revealed the following features of the enzyme. (i) The Mr = 148,000 alpha subunit is very likely derived by in vitro proteolysis from polypeptides with molecular weights of 185,000 and 166,000 that are present in vivo. (ii) The Mr = 60,000 beta subunit occurs in rapidly replicating embryos as both an 85,000- and a 60,000-dalton form, but predominantly as a 60,000-dalton form in more slowly replicating cultured cells. (iii) There is no detectable immunologic cross-reactivity between the four subunits. (iv) There is an abundance of antigenic material in embryos that co-migrates with the delta subunit of the purified enzyme during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

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.     

Cyanobacterial phycobilisomes. Role of the linker polypeptides in the assembly of phycocyanin

The phycocyanin-containing segments of the rod substructures of Anabaena variabilis phycobilisomes consist of complexes of phycocyanin with "linker" polypeptides of 27,000 and 32,500 daltons (Yu, M.-H., Glazer, A. N., and Williams, R. C. (1981) J. Biol. Chem. 256, 13130-13136). Complexes (alpha beta)3.27,000, (alpha beta)3.32,500, (alpha beta)6.27,000, [(alpha beta)6.32,500]n, (alpha beta)6.27,000 - (alpha beta)6.32,500 were prepared, where alpha beta represents a monomer of phycocyanin, and 27,000 and 32,500 represent the 27,000- and 32,500-dalton polypeptides, respectively. Tryptic digestion of (alpha beta)3.32,500 leads to a stable (alpha beta)3.28,000 complex which does not form higher aggregates. The 32,500 polypeptide is stable to trypsin in the [(alpha beta)6.32,500]n and (alpha beta)6.27,000 - [(alpha beta)6.32,500]n=1.2 aggregates. Upon trypsin treatment of all 27,000 still assembled into higher aggregates, (alpha beta)6.21,0900 and (alpha beta)6.21,000 - (alpha beta)6.32,500. The spectroscopic properties of phycocyanin-linker polypeptide complexes were not modified by the tryptic cleavages. These results show that the 32,500 polypeptide has two distinct functional domains, a 28,000 portion necessary to the stabilization of a trimeric phycocyanin complex and a 4,500 domain which links consecutive phycocyanin hexamers in the rod substructure. The 27,000 polypeptide likewise has two distinct functional domains: a 21,000 domain stabilizes a trimeric phycocyanin complex, a 6,000 domain is exposed in all of the assembly forms examined. From these and earlier studies, it is concluded that the 6,000 domain functions in the attachment of the rod substructures to the core of the phycobilisome.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Chemical evidence for the separation of G gamma and A gamma chains by polyacrylamide gel electrophoresis in urea and triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms

When outer-row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S alpha- and beta-heavy chains and the 83,000-dalton and 70,000-dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the alpha-chain and a 16,000-dalton light chain and between the two intermediate chains. Additional close associations of the beta-heavy chain with an 18,000-dalton light chain and 70,000-dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.     

The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans was inactivated by incubation with n-alkanols at 37 degrees C. The concentration of the alcohol required for complete inactivation decreased with increasing chain length; e.g. 2 M ethanol was as potent as 2 mM hexanol or 0.5 mM decanol. The data indicate a binding of the alcohol to the enzyme with an energy of about 4 kJ/methylene group. Sodium ions prevented the inactivation (50% at 30 mM NaCl). K+, NH4+, Cs+ and Mg2+ had no influence, whereas Li+ was ten times less effective than Na+. The enzyme was cleaved during the inactivation into a soluble part, consisting of the alpha (Mr 120,000) and beta polypeptide chains (60,000), whereas the hydrophobic gamma chain (30,000) precipitated. The soluble part catalysed the sodium-ion-independent but avidin-sensitive glutaconyl-CoA/crotonyl-CoA exchange as measured with the substrates [3-3H]crotonyl-CoA and unlabelled glutaconate and with glutaconate CoA-transferase as auxiliary enzyme. In the presence of free biotin or its methyl ester the soluble part catalysed the formation of crotonyl-CoA from glutaconyl-CoA (apparent Km for biotin 40 mM, Vmax 1% of the native decarboxylation reaction). This apparent reactivation was most likely caused by the carboxylation of free biotin. Based on these and other observations the following functions may be assigned to the different polypeptide chains of glutaconyl-CoA decarboxylase: biotin carrier (alpha), carboxytransferase (beta) and carboxylase, the actual sodium pump (gamma).     

DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression

The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule.