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R F Troxler

Publications and source records attributed to R F Troxler.

At least 91 records · Page 5Linked to original sources
PubMed1983

Phycobiliprotein synthesis in the unicellular rhodophyte, Cyanidium caldarium. Cell-free translation of the mRNAs for the alpha and beta subunit polypeptides of phycocyanin.

Phycobiliproteins are a class of abundant light-harvesting proteins assembled in complex multimeric aggregates located on thylakoid membranes of red algae and cyanobacteria. This study was undertaken to investigate the molecular basis of phycobiliprotein synthesis in the unicellular red alga, Cyanidium caldarium. RNA was isolated and separated into poly(A)-enriched and poly(A)-deficient fractions by oligo(dT)-cellulose chromatography. Both fractions stimulated incorporation of radiolabeled amino acids into protein in a reticulocyte lysate translation system. The alpha and beta subunit polypeptides of phycocyanin were among the products of poly(A)-deficient (but not poly(A)-enriched) RNA directed translation reactions on the basis of Mr and immunological cross-reactivity with immune serum prepared against native phycocyanin. After chromatography of post-oligo(dT)-cellulose poly(A)-deficient RNA on poly(U) agarose, the messenger RNAs for the alpha and beta subunit polypeptides of phycocyanin were again recovered in the poly(A)-deficient fraction, confirming that these messenger RNAs did not appear to be polyadenylated. Automated radiosequencing of in vitro synthesized alpha and beta subunit polypeptides of phycocyanin labeled with either [35S]methionine, [3H]isoleucine, or [3H]phenylalanine revealed partial amino acid sequences which were the same as the NH2-terminal sequences of native phycocyanin subunits. This demonstrates that a "transit peptide", such as that found in several proteins made in the cytosol and transported into chloroplasts, is not present on the subunits of phycocyanin.

Animals↗
PubMed1983

Cell-free translation of proline-rich protein mRNAS from human submandibular gland.

RNA was isolated from a human submandibular gland and separated into poly A-enriched and poly A-deficient fractions by chromatography on oligo (dT) cellulose. Both of these RNA fractions stimulated methionine incorporation into polypeptides in a reticulocyte lysate translation system. Two in vitro translation products templated by poly A-enriched mRNA were isolated by immunoprecipitation with immune serum directed against human salivary anionic proline-rich protein I. These polypeptides were shown to be precursors of proline-rich proteins on the basis of Mr, affinity for the antiserum, and preferential incorporation of proline. This study is the first to demonstrate cell-free translation of the mRNAs for human proline-rich salivary protein precursors.

Aged↗
PubMed1982

The effect of N-methylprotoporphyrin IX on the synthesis of photosynthetic pigments in Cyanidium caldarium. Further evidence for the role of haem in the biosynthesis of plant billins.

N-Methylprotoporphyrin IX strongly inhibits synthesis of phycocyanobilin, but not chlorophyll a, in the dark. In the light, both phycocyanin and chlorophyll a synthesis are inhibited in parallel. These results are consistent with the intermediacy of haem in algal bilin synthesis and suggest a control mechanism for chlorophyll a synthesis, previously unknown.

Aminolevulinic Acid↗
PubMed1982

Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide.

Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from pooled human collections displayed the same groups of proteins when fractionated by anion exchange and gel filtration chromatography. We have isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a proline-rich phosphopeptide (M-statherin) from macaque parotid saliva. MPRP has an apparent molecular weight of 16,900 and displays an unusual chemical composition. It is enriched in proline, glycine, and acidic amino acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25% (w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of protein. M-statherin is a 42-residue phosphopeptide with a high proline, glutamic acid, and tyrosine content, but which lacks threonine, valine, cysteine, methionine, isoleucine, and histidine. The complete covalent structure of M-statherin (Mr = 5,368) is: NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Arg-Tyr- -Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-Tyr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of a component in the salivary secretion of a subhuman primate. Phosphoserine occurs at residues 2 and 3. All 13 acidic and basic amino acids are located in the NH2-terminal half of the molecule. The carboxyl-terminal half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is repeated three times, the dipeptide Gln-Pro occurs twice, and the tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once. Evaluation of secondary structure by the Chou-Fasman method predicts an alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the molecule. Both MPRP and M-statherin inhibit spontaneous and seeded precipitation from solutions supersaturated with respect to calcium phosphate salts. This suggests that these macaque compounds may function by maintaining saliva supersaturated with respect to calcium phosphate salts, a necessary requirement for stabilization of hydroxyapatite in the surface layers of teeth.U

Amino Acid Sequence↗
PubMed1981

Primary structure of phycocyanin from the unicellular rhodophyte Cyanidium caldarium. II. Complete amino acid sequence of the beta subunit.

The complete amino acid sequence of the beta subunit of phycocyanin from the unicellular rhodophyte Cyanidium caldarium, has been determined by automated sequential degradation of cyanogen bromide, tryptic, and Staphylococcus aureus V8 protease peptides. The beta subunit contains 172 amino acids with methionine and glutamine the NH2- and carboxyl-terminal amino acids, respectively. The calculated molecular weight of the protein, based on the sequence, is 19,572. Two phycocyanobilin chromophores are covalently attached by cysteinyl thioether linkages to residues 82 and 153. A third cystine (residue 109) occurs in the beta subunit, but it is not attached to phycocyanobilin. Comparison of the complete amino acid sequence of the beta subunit of C. caldarium phycocyanin with the sequences of the phycocyanin beta subunits from two cyanobacteria, shows that the sequence homology previously noted at the NH2 terminus of phycobiliproteins from distantly related organisms extends along the entire polypeptide chain. The amino acid sequences of the alpha and beta subunits of C. caldarium phycocyanin are also similar, and by proper alignment of the sequences it can be shown that the beta subunit contains a 12-residue insertion where the second phycocyanobilin chromophore is covalently attached. A matrix comparing the alpha and beta subunits of phycobiliproteins for which the complete sequences are known has been determined, and based on these data, a scheme is proposed for evolution of the family of phycobiliproteins in living cyanobacteria and red algae from a protein precursor which gave rise initially to a beta-type allophycocyanin subunit.

Amino Acid Sequence↗
PubMed1981

Primary structure of phycocyanin from the unicellular rhodophyte Cyanidium caldarium. I. Complete amino acid sequence of the alpha subunit.

The complete amino acid sequence of the alpha subunit of phycocyanin from the unicellular rhodophyte Cyanidium caldarium has been determined by automated sequential degradation of cyanogen bromide peptides, tryptic peptides derived from protein chemically modified with 1,2-cyclohexanedione or citraconic anhydride, and a peptide obtained after cleavage of the protein at the single tryptophan residue. The alpha subunit contains 162 amino acids and methionine and serine are the NH2- and carboxyl-terminal amino acids, respectively. The calculated molecular weight of the protein, based on the amino acid sequence, is 18,303, in good agreement with the value of 17,500 +/- 500, obtained by electrophoresis on calibrated sodium dodecyl sulfate-polyacrylamide gels. One phycocyanobilin chromophore is attached to the alpha subunit at residue 84 by a cysteinyl thioether linkage. A second cysteine (residue 98) is present but is not linked to phycocyanobilin. The amino acid sequence of the alpha subunit of phycocyanin from C. caldarium is the first complete amino acid sequence of a phycobiliprotein from a eukaryotic alga. Extensive homology occurs between the alpha subunit of phycocyanin from C. caldarium and from two prokaryotic cyanobacteria, and the significance of this is discussed.

Amino Acid Sequence↗
PubMed1981

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.

Aminolevulinic Acid↗
PubMed1980

Allophycocyanin from Nostoc sp. phycobilisomes. Properties and amino acid sequence at the NH2 terminus of the alpha and beta subunits of allophycocyanins I, II, and III.

Allophycocyanin from Nostoc sp. phycobilisomes was separated into four spectrally distinct components designated allophycocyanin I, B, II, and III by adsorption chromatography on brushite columns. Allophycocyanins I and B had red-shifted fluorescence emission maxima, and on this basis, may function in transfer of excitation energy from phycobilisomes to chlorophyll a. Allophycyanins II and III, which together comprise 70% of the total allophycocyanin, have absorption maxima at 648 nm and 650 nm, respectively, and probably transfer excitation energy from phycocyanin to allophycocyanins I and B, in addition to serving a structural function. Allophycocyanin I was resolved into alpha, beta, and gamma subunits with apparent molecular weights of 18,000, 17,000, and 35,000, respectively, whereas allophycocyanin B was resolved into two subunits with apparent molecular weights of 16,100 and 15,300, using a modified Weber and Osborn gel electrophoresis system (Brown, A. S., and Troxler, R. F. (1977) Biochem. J. 163, 571-581). In the same gel system, allophycocyanins II and III were each resolved into alpha and beta subunits with apparent molecular weights of 18,000 and 17,000, respectively. The subunits of allophycocyanins I, II, and III were isolated by gel filtration and ion exchange chromatography and the amino acid compositions determined. Automated sequence analysis demonstrated that the first 30 amino acids at the NH2 terminus of alpha subunits, and the beta subunits, of allophycocyanins I to III were identical. alpha Subunits: Ser-Ile-Val-Thr-Lys-Ser-Ile-Val-Asn-Ala-Asp-Ala-Glu-Ala-Arg-Tyr-Leu-Ser-Pro-Gly -Glu-Leu-Asp-Arg-Ile-Lys-Ser-Phe-Val-Thr- beta Subunits: Ala-Gln-Asp-Ala-Ile-Thr-Ala-Val-Ile-Asn-Ala-Ala-Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp-Ala-Thr-Ala-Leu-Ser-Lys-Leu-Lys-Ala-Tyr- The gamma subunit of allophycocyanin I and both subunits of allophycocyanin B appeared to be blocked at the NH2 terminus, suggesting that the allophycocyanin B subunits may be different gene products than those of allophycocyanins I to III, or if the same, the subunits of allophycocyanin B undergo proteolytic modification after initial synthesis.

Amino Acid Sequence↗
PubMed1980

Mechanism of photooxidation of bacteriochlorophyll c derivatives. A possible model for natural chlorophyll breakdown.

The photooxidation of methyl pheophorbides, derived from bacteriochlorophyll c, yields ring-opened acetylbilitrienes which are structurally related to mammalian bile pigments. The mechanism of this photooxidation, and that of certain mesomethyl-substituted model compounds, has been studied using 18O labeling with molecular oxygen. In all cases, the methine bridge carbon atoms were retained in the ring-opened photoproducts and both oxygen atoms incorporated into the acetylbilitriene photoproducts were derived from a single oxygen molecule. In this respect, ring opening of bacteriochlorophyll c derivatives and mesomethyl-substituted model compounds differs from the nonphotochemical (enzymatic) ring opening of heme which involves elimination of the methine bridge carbon atom as carbon monoxide and insertion of two oxygen atoms, each from a different molecule of molecular oxygen. The process of photooxidation of bacteriochlorophyll c and mesomethyl-substituted model compounds studied may have implications for the mechanism of natural degradation of chlorophyll a and b during leaf senescence and grain ripening.

Bacteria↗
PubMed1980

Mechanism of bile-pigment synthesis in algae. 18O incorporation into phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium.

The origin of the lactam oxygen atoms of phycocyanobilin from Cyanidium caldarium was studied using 18O labelling. By inhibiting photosynthesis, a high 18O enrichment was maintained in the gas phase and the resulting incorporation of label showed that the lactam oxygen atoms were derived from two oxygen molecules. Slow exchange of these oxygen atoms with water was demonstrated directly by using H218O.

Bile Pigments↗
PubMed1978

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.

Apoproteins↗
PubMed1978

Phycocyanobilin synthesis in the unicellular rhodophyte Cyanidium caldarium.

Light is required for synthesis of the accessory photosynethetic pigment phycocyanin in cells of the unicellular rhodophyte Cyanidium caldarium. Phycocyanin is a conjugated protein composed of polypeptide subunits to which the light-absorbing bile pigment chromophore phycocyanobilin is covalently attached. Dark-grown cells of C. caldarium are unable to make phycocyanin, but when incubated in the dark with 5-aminolaevulinate the cells synthesize and excrete a protein-free phycobilin (algal bile pigment) into the suspending medium. The electronic absorption spectrum, electron impact mass spectrum, chromatographic properties and imide products obtained after chronic acid degradation of the excreted phycobilin were identical with those of phycocyanobilin cleaved from phycocyanin in boiling methanol. This establishes the structural identity between the excreted phycobilin, which is the end product of bile-pigment synthesis in vivo, and the chromophore cleaved from phycocyanin in boiling methanol. The significance of the structure of the excreted phycobilin with respect to the events surrounding the assembly of the phycocyanin molecule in vivo is discussed.

Bile Pigments↗