Search PubMed⌕ Search

Biomedical subjects

H Roy

Publications and source records attributed to H Roy.

At least 37 records · Page 2Linked to original sources
PubMed1989

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation.

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.

Journal Article↗
PubMed1988

Assembly of Rubisco from native subunits.

Large subunits of ribulosebisphosphate carboxylase/oxygenase (Rubisco) (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) from prokaryotic sources can assemble into intact enzyme either in vitro or in Escherichia coli cells. Large subunits of higher plant Rubisco do not assemble into Rubisco in E. coli cells, nor is it possible to reconstitute higher plant Rubisco from its dissociated subunits in vitro. This behavior represents an obstacle to any practical attempts at engineering the higher plant enzyme, and it suggests that the in vivo assembly mechanism of higher plant Rubisco must be more complex than is commonly expected for oligomeric proteins of organelles. In pea chloroplasts, a binding protein interacts with newly synthesized large subunits, in quantities expected for an intermediate in the assembly process, as judged by Western blotting. Radiotracer-labeled large subunits which interact with this binding protein can be shown to assemble into Rubisco in reactions which lead to changes in the aggregation state of the binding protein. Antibody to this binding protein specifically inhibits the assembly of these subunits into Rubisco. Rubisco synthesis appears to be subject to many types of control: gene dosage, transcription rate, selective translation of message, post-translational degradation and threshold concentration effects have been observed in various organisms' synthesis of Rubisco. The biochemical mechanisms underlying most of these effects have not been elucidated. The post-translational assembly mechanism in particular appears to require further study.

Adenosine Triphosphate↗
PubMed1988

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis.

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than ;mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.

Journal Article↗
PubMed1988

Stability and Dissociation of the Large Subunit RuBisCO Binding Protein Complex in Vitro and in Organello.

We are studying the stability of the binding protein which associates with newly synthesized large subunits of ribulose bisphosphate carboxylase. In chloroplast extracts, it has been shown that a dodecameric complex of the large subunit binding protein dissociates extensively into binding protein monomers and 7S (117 kilodaltons) large subunit-containing complexes in the presence of ATP. The concentrations of ATP which bring this about are quite low, prompting some investigators to suggest that the dodecameric complex might not exist in vivo. We have found, however, that in concentrated chloroplast extracts, at protein concentrations which are closer to those which occur in organello, the dissociation of the binding protein complex by ATP is much less extensive. For this reason, we have tested the stability of the binding protein in organello, by illuminating chloroplasts followed by lysis and polyacrylamide gel electrophoresis of the extracts. Radioactive large subunits associated with the dodecameric binding protein dissociated extensively in the light. The results are consistent with the idea that the high molecular weight form of the binding protein can function as a reservoir of large subunits which can be tapped in vivo, in a reaction dependent on light and ATP.

Journal Article↗
PubMed1986

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein.

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.

Adenosine Triphosphate↗
PubMed1984

ATP-released large subunits participate in the assembly of RuBP carboxylase.

Preincubation of 35S-methionine-labeled chloroplast extracts with ATP at 0 degree C potentiates the subsequent assembly of labeled large subunits into RuBPCase . This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide-stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of RuBPCase .

Adenosine Triphosphate↗
PubMed1983

Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase.

Ribulose-1,5-bisphosphate carboxylase [RuP(2)Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is composed of eight small subunits (M(r), 14,000) and eight large subunits (M(r), 55,000). Newly synthesized large subunits are associated with two complexes having sedimentation coefficients of 7 and 29 S. Assembly of RuP(2)Case occurs in isolated intact chloroplasts in the light but not in the dark. When extracts of chloroplasts are treated with ATP or GTP, RuP(2)Case assembly is accelerated while the 29S large subunit complex is maintained. In the presence of Mg(2+), ATP brings about almost complete dissociation of the 29S complex, whereas GTP and a nonhydrolyzable analog of ATP are without effect. These results indicate the existence of a complex set of reactions involving nucleotides, Mg(2+), and several putative intermediates in RuP(2)Case assembly. It is postulated that these reactions at least partly account for the light dependence of RuP(2)Case assembly. In particular, ATP and GTP promote the assembly of large subunits into RuP(2)Case.

Journal Article↗
PubMed1982

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts.

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.

Carboxy-Lyases↗
PubMed1978

Small subunit contacts in ribulose-1,5-bisphosphate carboxylase.

The arrangement of subunits of ribulosebisphosphate carboxylase in solution has been studied by exposing the enzyme to the cross-linking agents tetranitromethane, dimethyl suberimidate, and dimethyl adipimidate, and the cleavable cross-linking agent, methyl 4-mercaptobutyrimidate followed by gel electrophoresis in the presence of dodecyl sulfate. All these agents caused the formation of dimers of the enzyme's small subunit, independently of protein concentration. In addition, trimers and tetramers of small subunit were detected in the mercaptobutyrimidate-treated enzyme. The data show that small subunits are closely paired in the native enzyme and may be in layers of four, or a ring of eight.

Carboxy-Lyases↗
PubMed1977

Synthesis of the Small Subunit of Ribulose-1,5-bisphosphate Carboxylase by Soluble Fraction Polyribosomes of Pea Leaves.

The products of amino acid incorporation by pea (Pisum sativum L.) leaf soluble fraction polyribosomes in the wheat germ system were examined by two-dimensional electrophoresis and fluorography.There are two isoelectric variants of the small subunit of ribulose bisphosphate carboxylase in this organism, and the more alkaline of these is consistently labeled in the cell-free protein-synthesizing system. The more acid variant is also labeled, but often less extensively. A minority of leaf polyribosomes are recovered from low speed sedimentable fractions. While these appear also to synthesize the small subunits, the patterns of labeling do not indicate a preferential synthesis of these polypeptides by the sedimentable fraction polyribosomes.In the same experiments, labeling of the large subunit spots was sharply below background; these results confirm a cytoplasmic site of synthesis for small subunits of ribulose bisphosphate carboxylase.

Journal Article↗