Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.     

Ethanol production from xylose with a pyruvate-formate-lyase mutant of Klebsiella planticola carrying a pyruvate-decarboxylase gene from Zymomonas mobilis

Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO₂ and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO₂. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

The primary structure of cytochrome c from the nematode Caenorhabditis elegans.

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an 'invariant' phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.     

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.     

Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

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.