The Amino Acid Composition of Human Pituitary Growth Hormone

The amino acid content in human hypophyseal growth hormone has been determined by chromatography on resin columns. On the basis of 29,000 for the molecular weight, the empirical formula of the hormone was obtained: lys₁₃his₅arg₁₄asp₂₇ thr₁₄ser₂₃glu₃₄pro₁₂gly₁₃ala₁₂-(1/2 cys)₆val₁₂met₄-ileu₁₀leu₃₁tyr₁₀phe₁₅try₁(NH₃)₃₂.     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’     

Platyhelminth mitochondrial DNA: Evidence for early evolutionary origin of a tRNAserAGN that contains a dihydrouridine arm replacement loop,and of serine-specifying AGA and AGG codons

Summary The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the liver flukeFasciola hepatica (phylum Platyhelminthes, class Trematoda) has been determined, within which have been identified the genes for tRNA^ala, tRNA^asp, respiratory chain NADH dehydrogenase subunit I (ND1), tRNA^asn, tRNA^pro, tRNA^ile, tRNA^lys, ND3, tRNA^serAGN, tRNA^trp, and cytochromec oxidase subunit I (COI). The 11 genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of theF. hepatica mitochondrial genes differs from what is found in other metazoan mtDNAs. All of the sequenced tRNA genes except the one for tRNA^serAGN can be folded into a secondary structure with four arms resembling most other metazoan mitochondrial tRNAs, rather than the tRNAs that contain a TψC arm replacement loop, found in nematode mtDNAs. TheF. hepatica mitochondrial tRNA^serAGN gene contains a dihydrouridine arm replacement loop, as is the case in all other metazoan mtDNAs examined to date. AGA and AGG are found in theF. hepatica mitochondrial protein genes and both codons appear to specify serine. These findings concerningF. hepatica mtDNA indicate that both a dihydrouridine arm replacement loop-containing tRNA^serAGN gene and the use of AGA and AGG codons to specify serine must first have occurred very early in, or before, the evolution of metazoa.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Immunogold localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in the nitrogen-fixing cyanobacterium,Plectonema boryanum

Summary Antiserum against the Calvin cycle enzyme, ribulose-1,5-bisphosphate carobxylase/oxygenase (RuBisCO), was used in conjunction with colloidal gold to localize RuBisCO in nitrogen-fixing (fix+) and nonfixing (fix–)Plectonema boryanum cells. RuBisCO antiserum consistently labeled the cytoplasm and polyhedral bodies (carboxysomes) in both fix+ and fix– cells. Through morphometry, it was determined that significantly less gold label (indicative of RuBisCO) was present in fix+ cells. This decreased RuBisCO content correlated with a decrease in net photosynthetic oxygen evolution also observed in fix+P. boryanum.Abbreviations RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase - fix+ nitrogen-fixing - fix– nonfixing     

The mutagenic effect of ultraviolet radiation and nitrous acid onMycobacterium phlei

Lethal and mutagenic effects of nitrous acid and UV radiation onMycobacterium phlei were studied Three auxotrophic strains of the PA strain ofMycobacterium phlei were obtained: ala^-, his^-, and gly^- (ser^-) INH^r Bods of the his^- strain grown in liquid media are longer to filamentous as compared with cells of the prototrophic PA strain grown in the same media, whereas cells of the gly^- (ser^-) INH^r mutant are shorter to coccobacillary. Cells of the ala^- strain are characterized by their various length from normal to coccobacillary. The auxotrophic strains obtained differ from each other by a frequency of spontaneous reversions to prototrophy. The his^- strain is the most stable, a frequency of spontaneous reversions to prototrophy being 10^-9. The gly^- (ser^-) INH^r strain reverts spontaneously to prototrophy with a frequency of 10^-8 to 10^-7. The ala^- strain spontaneously reverting with a frequency of 10^-5 is the most labile. The auxotrophic mutants obtained do not differ from the original prototrophic strain in the other properties studied. A change in a frequency of INH and STM-resistant mutants was also studied. It was found that under the influence of UV radiation a frequency of INH-resistant mutants increases 43 to 80 fold as compared with a frequency of spontaneous mutations, this latter being about 2.6 × 10^-6. No substantial increase in a frequency of STM-resistant mutants was found using UV irradiation under the given experimental conditions; their spontaneous frequency equals to 9.0 × 10^-9 to 2.0 X 10^-8.     

Glyceraldehyde 3-P dehydrogenase, glycerate 3-P kinase and enolase mutants of Escherichia coli: Genetic studies

Summary The genetic loci for two enzymes of glycolysis have been established by transductional crosses. The eno locus, likely to be the structural gene for enolase maps in the 52-minute region of the Escherichia coli chromosome. A structural determinant for glycerate 3-P kinase (pgk) is located near serA. The map order is speB-pgk–serA-lysA-argA-eno-cysC. In the 35-minute region maps a locus affecting the structure of glyceraldehyde 3-P dehydrogenase.     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Peptide chain elongation: A possible role of montmorillonite in prebiotic synthesis of protein precursors

Several studies have proven the ability of montmorillonite to catalyse amino acid condensation under assumed prebiotic conditions, simulating wetting-drying cycles. In this work, the oligomerization of short peptides gly₂, gly₃, gly₄ and ala₂ on Ca-and Cu-montmorillonite in drying-wetting cycles at 80 °C was studied. The catalytic effect of montmorillonite was found to be much higher than in the case of glycine oligomerization. From gly₂ after 3 weeks, 10% oligomers (up to gly6, with gly₃ as main products) are formed. Gly₃ and gly₄ give higher oligomers even after 1 cycle. Ala₂ produces both ala₃ and ala₄, whereas ala does not produce any oligomers under these conditions. Heteroologomerization was observed: ala-gly-gly is formed from ala and gly₂. Much higher yields are obtained using Ca-montmorillonite, because copper (II) oxidizes organic molecules. The influence of the reaction mechanism on the preferential oligomerization of oligopeptides is discussed.     

Quantum chemical modeling of the kinetic isotope effect of the carboxylation step in RuBisCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most important enzyme for the assimilation of carbon into biomass, features a well-known isotope effect with regards to the CO₂ carbon atom. This kinetic isotope effect α = k ₁₂/k ₁₃ for the carboxylation step of the RuBisCO reaction sequence, and its microscopic origin, was investigated with the help of cluster models and quantum chemical methods [B3LYP/6-31G(d,p)]. We use a recently proposed model for the RuBisCO active site, in which a water molecule remains close to the reaction center during carboxylation of ribulose-1,5-bisphosphate [B. Kannappan, J.E. Gready, J. Am. Chem. Soc. 130 (2008), 15063]. Alternative active-site models and/or computational approaches were also tested. An isotope effect alpha for carboxylation is found, which is reasonably close to the one measured for the overall reaction, and which originates from a simple frequency shift of the bending vibration of ¹²CO₂ compared to ¹³CO₂. The latter is the dominant mode for the product formation at the transition state.     

Amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from Euglena

Summary The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10^-9 per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.Abbreviations DABITC Dimethyl amino azobenzene isothiocyanate - HPLC high pressure liquid chromatography - Kd Kilo daltons - LSU large subunit - PITC phenyl isothiocyanate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - SSU small subunit - TFA trifluoric acetic acid     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.     

Isolation of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum

The enzyme ribulose bisphosphate carboxylase/oxygenase has been purified from Chromatium vinosum. When an extract is subjected to centrifugation at 35,000xg in the presence of polyethylene glycol (PEG)-6000 and the supernatant is treated with 50 mM Mg²⁺ and the precipitate is then fractionated by vertical centrifugation into a reoriented sucrose gradient followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A50, the resultant enzyme contains large (L) and small (S) subunits. Alternatively, centrifugation of extracts at 175,000xg in the presence of PEG-6000 followed by fractionation with Mg²⁺, density gradient centrifugation, and chromatography on DEAE-Sephadex A50 yields an enzyme free of small subunits. The two forms have comparable carboxylase and oxygenase activities and have compositions and molecular weights corresponding to L₈ and L₈S₈ enzymes. The amino acid compositions of L and S subunits are reported. The L₈S₈ enzyme from spinach cannot be similarly dissociated by centrifugation at 175,000xg in the presence of PEG-6000.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine-tetraacetate - MOPS 3-(N-morpholino)propanesulfonic acid - PEG polyethylene glycol - RuBisCO d-ribulose 1,5-bisphosphate caboxylase/oxygenase - RnBP d-ribulose 1,5-bisphosphate - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor G. Drews on occasion of his 60th birthday     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Effects of Photosynthetic Intermediates on the Activation State of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z

The half-saturating concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Euglena gracilis Z for CO₂ in its activation by CO₂ in the presence of a saturating concentration of MgCl₂ (KJ was measured by analyzing the partial reversible inactivation of the fully activated enzyme in the medium with dilute CO₂. The K_d of the Euglena enzyme was 12.5 μm. The K,_d values were 6.3/im for the enzyme from soybean, 10.8 fiM from maize, 23.3 jiM from Scenedesmus obliquus, and 20.8 μm from Anabaena 7120. The activated state of Euglena RuBisCO was stabilized by 6-phosphogluconate, fructose 1,6-bisphosphate, and 3-phosphoglycerate in the medium containing low concentrations of CO₂. Both fructose 6-phosphate and ATP stimulated inactivation in the medium. NADPH not only stabilized the activated state of the enzyme, but also enhanced the enzyme activity over the full activity measured in the absence of NADPH. NADP⁺ did not nullify the effects of NADPH on the activation at all. The physiological significance of the effects of these photosynthetic metabolites on the activated state of Euglena RuBisCO is discussed.     

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate

Homogeneous D-ribulose 1,5-bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, $\text{Chlamydomonas reinhardtii}$, and $\text{Hydrogenomonas eutropha}$ are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from $\text{Rhodospirillum rubrum}$, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from $\text{Rhodospirillum rubrum}$.     

Informational content of mitochondrial DNA from a "low density" petite mutant of yeast

Mitochondrial DNA from wild-type yeast $\text{Saccharomyces cerevisiae}$ contains the genetic information for some mitochondrial tRNAs including tRNA_ser and tRNA_phe. In a “low density” petite mutant, mitochondrial DNA still retains the information for tRNA_ser, while the information for tRNA_phe is lost.The permanence of genetic information in this DNA containing only 3.6% G+C supports previous results concerning its intramolecular heterogeneity. An irregular distribution of G+C content along the molecule was further demonstrated by annealing experiments performed with DNA fragmented by sonication and fractionated on CsCl density gradient. These experiments show that the heavy fractions of the gradient preferentially anneal with mitochondrial seryl-tRNA.