Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Characterisation of neuropeptides of the AKH/RPCH-family from corpora cardiaca of Coleoptera

Summary Extracts of corpora cardiaca from two members of the family Tenebrionidae,Zophobas rugipes andTenebrio molitor, from one member of the Chrysomelidae,Leptinotarsa decemlineata, and from three members of the Scarabaeidae,Pachnoda marginata, P. sinuata andMelolontha hippocastani, were assayed for adipokinetic and hypertrehalosaemic activity in acceptor locusts (Locusta migratoria) and cockroaches (Periplaneta americana), respectively. All corpus cardiacum material tested, except that from the cockchafer,M. hippocastani, gave positive bioassay results. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high performance liquid chromatography (RP-HPLC). Gland extracts from the two tenebrionid species each show a single peak of biological activity associated with a single peak of UV absorbance having an identical retention time in both species. The two biologically active fractions from the corpora cardiaca of the potato beetle,L. decemlineata, coelute with exogenous (synthetic) hypertrehalosaemic hormones I and II of the American cockroach. The two species of the genusPachnoda contain two active compounds in their glands; compound I of each species is more abundant and elutes just ahead of the (synthetic) hypertrehalosaemic hormone of the cockroachBlaberus discoidalis. The gland material ofM. hippocastani exhibits and absorbance peak with the same retention time as the major peak from thePachnoda-species; however, this peak material does not elicit biological activity in the assays used here. After fractionation by RP-HPLC the main biologically active compounds were subjected to amino acid analyses. All factors are peptidic and contain 8 amino acid residues. The peptides from the tenebrionid species have the amino acid residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp_(i), whereas the main peptide from corpora cardiaca ofP. marginata contains the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Tyr₍₁₎, Leu₍₁₎ and Trp₍₁₎. Amino acid composition analyses of the two active fractions fromL. decemlineata reveal the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Val₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound I and Asx₍₁₎, Glx₍₁₎, Thr₍₂₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound II.     

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)     

The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17

Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.     

Genetics of the quantitative Lp(a) lipoprotein trait

The Lp(a) lipoprotein is a complex particle composed of a low density lipoprotein (LDL)-like lipoprotein and the disulfide bonded Lp(a) glycoprotein. The complex represents a quantitative genetic trait. SDS gel electrophoresis under reducing conditions of sera followed by immunoblotting with affinity-purified polyclonal anti-Lp(a) demonstrated inter- and intra-individual size heterogeneity of the glycoprotein with apparent Mr in the range 400-700kDa. According to their relative mobilities compared to apo B-100 the Lp(a) patterns were categorized into phenotypes F, B, S1, S2, S3 und S4 and into the respective double-band phenotypes. This size heterogeneity seems to be controlled by multiple alleles designated LpF, LpB, LpS1, LpS2, LpS3, LpS4 and a null allele (LpO) at a single locus. Phenotype frequencies observed in 441 unrelated subjects were in good agreement with those expected from the genetic hypothesis. Comparison of Lp(a) lipoprotein concentrations in the different phenotypes revealed a highly significant association of phenotypes B, S1 and S2 with high, and phenotypes S3 und S4 with intermediate Lp(a) concentrations. A third mode is represented by the null phenotype were no Lp(a) band is detected upon immunoblotting and Lp(a) lipoprotein is low or absent. We conclude that the same gene locus is involved in determining Lp(a) glycoprotein phenotype and Lp(a) lipoprotein concentrations in plasma. This major gene seems to be the Lp(a) glycoprotein structural gene locus.