A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Transient states of geosmin,pigments, carbohydrates and proteins in continuous cultures of Oscillatoria brevis induced by changes in nitrogen supply

Transitions in the growth limiting factor from light (I) to nitrogen (N) and vice versa caused changes in geosmin production, protein and carbohydrate content, and the synthesis of pigments such as chlorophyll a (Chl a), phycobiliproteins (PBPs), and -carotene of the cyanobacterium Oscillatoria brevis. Following IN transition the first 150h, the decrease in protein content was compensated for by an increase of carbohydrates, and thereby, a constant biomass level was maintained in this period. Thereafter, biimass dropped to 15% of its initial level. A decrease in geosmin and pigment content was observed during transition from IN-limited growth. However, geosmin increased relative to phytol (Chl a) and -carotene which may indicate that a lowered demand for phytol and -carotene during N-limited growth allows isoprenoid precursors to be directed to geosmin rather than to pigment synthesis. Synthesis of Chl a and -carotene at the expense of geosmin was suggested for the observed start of increase in geosmin production only at the time that Chl a and -carotene had reached their I-limited steady state. Transition from nitrogen to light limited growth caused an acceleration of metabolism shown by a rapid decrease in carbohydrate content accompanied by an increase in protein content. The growth rate of the organisms temporarily exceeded the dilution rate of the culture and the biomass level increased 6-fold. Due to the only modest changes in geosmin production (2-fold) compared to changes in biomass level (6-fold) during I-or N-limited growth, environmental factors seem to have limited effect on geosmin production.Abbreviations Chl a chlorophyll a - dry wt dry weight; - I-limited light-limited - N-limited nitrogen-limited - PBP phycobiliprotein This research was performed at the Department of Microbiology, University of Amsterdam, with finacial support provided by the Royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Structural studies on cyanobacterial photosystem I

The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent M_r of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the M_r 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the M_r 17,700 polypeptide and of 42 residues for the M_r 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI photosystem I - P700 reaction center of PSI - SDS sodium dodecylsulfate - TBS tris-buffered saline - TTBS TBS containing Tween-20     

Purification and characterisation of an inducible β-galactosidase from Corynebacterium murisepticum

The -galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The K _m values of the enzyme for the substrates lactose and o-nitrophenyl--d-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

Organization and structure of Volvox beta-tubulin genes

Summary Genomic clones encoding two Volvox -tubulin genes have been isolated and shown to represent the only two -tubulin genes in the genome. Restriction fragment length polymorphism analysis was used to demonstrate that the two genes are genetically linked. One of these genes was sequenced and the mRNA start site(s) determined by primer extension. A comparison of its sequence to those of the two -tubulin genes of Chlamydomonas revealed: (1) a high degree of conservation of the coding region, with the predicted amino acid sequence differing only in the C-terminal residue; (2) extensive sequence conservation in the 5 untranslated leader region and a 16 bp (putative regulatory) sequence in the promoter region; (3) the same number and location of introns, with a short region of homology in intron 1, but little significant homology in introns 2 and 3.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.