PubMed Health⌕ Search

Biomedical subjects

G W Schmidt

Publications and source records attributed to G W Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Immunochemical analysis of baboon (Papio cynocephalus) IgG subclasses.

Few reports are presently available on the existence of IgG subclasses in nonhuman primates. Papain and trypsin digestion of baboon (Papio cynocephalus cynocephalus and P. cynocephalus hamadryas) IgG proteins, in the absence of cysteine, revealed the occurrence of two different protein populations, one being protease-resistant, the other being protease-sensitive. The papain-resistant population is rendered sensitive to digestion upon addition of cysteine. Ion exchange chromatography of the papain-resistant IgG population subsequently demonstrated that it is composed of two different subpopulations varying in their ionic binding affinities. Peptide maps of the Fc fragments of the papain sensitive population and of the Fc fragments of the two papain-resistant subpopulations differing in their binding affinities for ion exchange resins, were different from each other. The biochemical identification of the baboon IgG proteins presented here strongly suggests that they are composed of at least three different subclasses.

Animals↗
PubMed1987

Reconstitution of chlorophyll a/b light-harvesting complexes: Xanthophyll-dependent assembly and energy transfer.

A method for in vitro reconstitution of the chlorophyll a/b light-harvesting complex from LiDodSO(4)/heat-denatured or acetone-extracted photosynthetic membranes has been developed. Characterization of the minimum components necessary for the functional organization of pigments in these membrane complexes reveals that xanthophylls are essential structural components.

Journal Article↗
PubMed1987

Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants.

Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O(2)-evolving PSII reaction center complex.

Journal Article↗
PubMed1986

Specific heat shock proteins are transported into chloroplasts.

We demonstrate that in three plant species-soybean, pea, and corn-certain nuclear-encoded heat shock proteins are transported into chloroplasts. In vitro translation products of poly(A)-RNA from control or heat-shocked plants were incubated with isolated intact pea chloroplasts and differences in the profile of imported proteins were analyzed. In all three species, abundant polypeptides between 21 and 27 kDa are present in the heat shock sample and absent in the controls. These polypeptides are protected from trypsin and chymotrypsin digestion after their import into chloroplasts and are recovered primarily with the soluble chloroplast protein fraction. Chloroplasts isolated from pea or corn leaves labeled in vivo at heat shock temperatures, but not at normal growth temperatures, contain the same polypeptides observed in vitro. Synthesis of the heat shock polypeptides can be inhibited in vivo by cycloheximide but not by chloramphenicol, further indicating they are products of cytoplasmic protein synthesis. The in vitro transport experiments demonstrate that synthesis of the chloroplast-localized heat shock proteins results from heat-induced accumulation of the corresponding poly(A)-RNAs. The same mRNAs are also produced in response to heat shock by a nonphotosynthetic tissue, the etiolated soybean hypocotyl.

Journal Article↗
PubMed1986

Biogenesis of photosystem II complexes: transcriptional, translational, and posttranslational regulation.

The integral membrane proteins of photosystem II (PS II) reaction center complexes are encoded by chloroplast genomes. These proteins are absent from thylakoids of PS II mutants of algae and vascular plants as a result of either chloroplast or nuclear gene mutations. To resolve the molecular basis for the concurrent absence of the PS II polypeptides, protein synthesis rates and mRNA levels were measured in mutants of Chlamydomonas reinhardtii that lack PS II. The analyses show that one nuclear gene product regulates the levels of transcripts from the chloroplast gene encoding the 51-kD chlorophyll a-binding polypeptide (polypeptide 5) but is not involved in the synthesis of other chloroplast mRNAs. Another nuclear product is specifically required for translation of mRNA encoding the 32-34-kD polypeptide, D1. The absence of either D1 or polypeptide 5 does not eliminate the synthesis and thylakoid insertion of two other integral membrane proteins of PS II, the chlorophyll a-binding polypeptide of 46 kD (polypeptide 6) and the 30-kD "D1-like" protein, D2. However, these two unassembled subunits cannot be properly processed and/or are degraded in the mutants even though they reside in the membrane. In addition, pulse labeling of the nuclear mutants and a chloroplast mutant that does not synthesize D1 mRNA indicates that synthesis of polypeptide 5 and D1 is coordinated at the translational level. A model is presented to explain how absence of one of the two proteins could lead to translational arrest of the other.

Chlamydomonas↗
PubMed1986

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations.

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.

Journal Article↗
PubMed1986

The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants.

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl(2) washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O(2) evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.

Journal Article↗
PubMed1985

Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5-bisphosphate carboxylase small subunit of Chlamydomonas.

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.

Amino Acid Sequence↗
PubMed1983

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate.

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.

Chlamydomonas↗
PubMed1983

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts.

We have detected a proteolytic mechanism in chloroplasts that selectively and rapidly degrades the imported small subunit of ribulose 1,5-bisphosphate carboxylase when pools of the chloroplast-synthesized large subunit are depleted. This degradation system is constitutively present and appears to be responsible for precise stoichiometric accumulation of the two subunits of the enzyme. We believe similar proteolytic mechanisms participate in regulating the accumulation of other photosynthetic proteins during chloroplast biogenesis.

Journal Article↗
PubMed1983

Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in Chlamydomonas reinhardtii.

The coordinated synthesis of the subunits of ribulose 1,5-bisphosphate carboxylase was examined by analysis of the chloroplast ribosome-deficient mutant of Chlamydomonas reinhardtii, ac20crl. The absence of the chloroplast-synthesized large subunit of this enzyme from cells of this strain is a direct consequence of the lack of chloroplast ribosomes. In contrast, the absence of the cytoplasmically-synthesized small subunit of ribulose 1,5-bisphosphate carboxylase from this mutant is not understood. To discern the cause of this absence, we have compared results of in vivo radioactive labeling experiments with those of cell-free translations of RNA from ac20crl. Protein products from these experiments were identified by one-and two-dimensional electrophoretic analyses. Neither subunit, revealed either as a stained band or by fluorography of proteins radioactively labeled in vivo for 2 hours, was detected in ac20crl. Cell-free translation of polyadenylated RNA obtained from ac20crl, however, revealed wild-type levels of mRNA for the precursor to the small subunit. This messenger was found to be associated with subribosomal RNP and polysomes. We conclude that the absence of the small subunit of ribulose 1,5-bisphosphate carboxylase from ac20crl is the result of a translational or posttranslational event.

Journal Article↗
PubMed1982

Optimal conditions for post-translational uptake of proteins by isolated chloroplasts. In vitro synthesis and transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase.

Many polypeptides translated in the cytosol enter the chloroplast where they assemble into macromolecular complexes. The transport of these polypeptides into the plastid can be examined in vitro by mixing isolated chloroplasts with pea poly(A) RNA translation products. Following optimization of both translation in the wheat germ system and the conditions during in vitro uptake, we observe the post-translational transport of over 100 polypeptides; many remain in the soluble phase of the organelle while others integrate into the thylakoid membranes. Most products transported in vitro co-migrate with in vivo products on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, with the improved conditions, we demonstrate the transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase into isolated plastids. While we have not been able to detect any cell-free translation product that is immunologically related to fructose-1,6-bisphosphatase, both plastocyanin and ferredoxin-NADP+ oxidoreductase are synthesized as precursors in vitro. These precursors are imported into the organelle where they are processed to the size of their mature counterparts. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the precursor to plastocyanin is 15,000 larger than the mature product and the precursor to ferredoxin-NADP+ oxidoreductase is 8,000 larger than the mature product.

Biological Transport↗
PubMed1981

Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Biological Transport↗
PubMed1979

NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii.

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro-synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.

Amino Acid Sequence↗