Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits

An expression plasmid incorporating the structural gene for the large subunit of a cyanobacterial ribulose-bisphosphate carboxylase, but not the gene for its complementary small subunit, directs the synthesis of large subunits in Escherichia coli. This provides a means for obtaining a preparation of large subunits completely devoid of small subunits, which is not otherwise achievable. In extracts, these large subunits were found predominantly in the form of octamers, but intersubunit interactions were weaker than in the holoenzyme, which contains eight small subunits as well as eight large subunits, and tended to be broken by procedures which separated octamers from lower oligomers and monomers. However, partial purification by anion-exchange chromatography was possible. The large subunits recognized the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate, thus enabling measurement of catalytic site concentrations, but the binding was much weaker than to the holoenzyme. E. coli-produced large subunits catalyzed carboxylation with a kcat of 1% of that of the holoenzyme and the substrate affinities were 3- to 5-fold weaker. They also assembled with heterologous small subunits isolated from spinach ribulose-P2 carboxylase with a 100-fold increase in catalytic activity under standard assay conditions. Since catalysis can proceed in their absence, the small subunits cannot be directly involved in the catalytic chemistry. Their stimulative influence upon catalysis must be exerted by conformational means.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

Fractional synthesis rates in vivo of skeletal-muscle myosin isoenzymes.

The synthesis rates of different myosin isoenzymes in a single muscle, and of the same isoenzymes in different muscles (soleus, masseter and plantaris), were measured. The rate of total protein synthesis was significantly higher in the soleus [greater than 95% slow myosin (SM)] than in the plantaris [greater than 95% fast myosin (FM)]. Two fast isoenzymes, FM2 and FM3, were synthesized at different rates in the masseter, and SM was synthesized at a faster rate than FM. Intermediate myosin had a synthesis rate similar to that of FM. There was a small but significant difference between the synthesis rates of the SM isoenzymes of the soleus and masseter muscles. FM3 was synthesized faster in the masseter than in the plantaris, whereas FM2 was synthesized faster in the plantaris than in the masseter.     

Evidence for two subclasses of methylamine dehydrogenases with distinct large subunits and conserved PQQ-bearing small subunits

Antibodies, which were raised to the isolated subunits of the methylamine dehydrogenases of Paracoccus denitrificans and bacterium W3A1, were used to demonstrate immunological cross-reactivity between the small pyrrolo-quinoline quinone bearing subunits of these two enzymes. The small subunits of these enzymes also exhibited nearly identical pI values. Conversely, the large subunits of the P. denitrificans and bacterium W3A1 enzymes did not cross-react immunologically and exhibited, respectively, very acidic and very basic pI values which reflected the electrostatic properties of their respective holoenzymes. Antibodies specific for the large subunit of the P. denitrificans enzyme, but not the bacterium W3A1 enzyme, cross-reacted with the analogous proteins in cell extracts of Thiobacillus versutus and Methylobacterium sp strain AM1.     

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.     

Ribosome structure determined by electron microscopy of Escherichia coli small subunits,large subunits and monomeric ribosomes

Unique, three-dimensional structures have been determined for Escherichia coli small subunits, large Subunits and monomeric ribosomes by electron microscopy of ribosomes and subunits and of antibody-labeled ribosomes and subunits.Small subunits orient on the carbon substrate with their long axes parallel to the plane of the carbon. In these projections the subunit is divided into a onethird and a two-thirds portion by a region of accumulated negative stain similar to that observed in eukaryotic small subunits. Four characteristic views, or projections, are readily recognized and correspond to orientations of approximately ?40 °, 0 °, +50 ° and +110 ° about the long axis of the subunit. Three of these have been described (Lake et al., 1974a; Lake & Kahan, 1975). The two most distinctive views are a quasi-symmetric view (0 °) that is characterized by an approximate line of mirror symmetry that coincides with the long axis of the subunit, and an asymmetric view (110 °) that is characterized by a concave and a convex subunit boundary. In the asymmetric projection, a platform or ledge is viewed “face-on”. The platform is attached to the lower two-thirds of the subunit just below the one-third/two-thirds partition. It is separated from the upper one-third of the subunit at the level of the partition and above the partition it forms a cleft approximately 30 to 40 Å wide, which has been suggested as the site of the codon-anticodon interaction (Lake & Kahan, 1975).Four characteristic views are presented for the large subunit. The most prominent of these, the quasi-symmetric view (θ = 90 °, φ = 0 °), is distinguished by a central protuberance located on a line of approximate mirror symmetry. The central protuberance is surrounded by projecting features inclined at about 50 ° on both sides of it. The smaller of these projections is rod-like, about 40 Å wide and approximately 100 Å long. The feature projecting from the other side of the central protuberance is shorter, more blunt and wider than the rod-like appendage. In another view approximately orthogonal to the quasi-symmetric projection, the asymmetric projection (θ = 10 °, φ = 90 °), the subunit profile is distinguished by a convex lower edge and an upper boundary which is indented by a notch. The subunit is separated, in projection, by the notch into two unequal regions. The smaller region comprises about 20% of the total projected density and consists of the central protuberance and the rod-like appendage.The profiles observed in fields of monomeric 70 S ribosomes result from superpositions of the 30 S and 50 S profiles. Two major views are observed, an overlap and a non-overlap view, corresponding to whether or not the profile of the small subunit overlaps that of the large subunit in the 70 S profile. The small subunit is oriented in the monomeric ribosome so that the platform is in contact with the large subunit. The central protuberance of the large subunit overlaps part of the upper one-third of the small subunit in the overlap view of 70 S ribosomes, although in three dimensions they are probably separated by 30 to 50 Å. A region of the small subunit comprising the platform, the cleft and part of the upper one-third, suggested to be the approximate binding site of IF3 and IF2 (Lake & Kalian, 1975), is located at the interface between the large and small subunits, in a region of the small subunit that is close to, but probably not in physical contact with, the large subunit.     

Three partial reactions of ribulose-bisphosphate carboxylase require both large and small subunits

Three partial reactions of ribulose-bisphosphate carboxylase/oxygenase were measured in the presence and absence of small subunits using the enzyme from the cyanobacterium, Synechococcus ACMM 323, whose small subunits may be reversibly dissociated from its octameric, large-subunit core. These partial reactions were: the exchange of the proton at C-3 of the substrate, ribulose 1,5-bisphosphate, with the medium which is indicative of C-2, C-3 enolization; the hydrolysis of the 6-carbon reaction intermediate, 3-keto-2-carboxy-D-arabinitol 1,5-bisphosphate, to two molecules of 3-phosphoglycerate; and the decarboxylation of the 6-carbon intermediate, which is catalyzed only by the deactivated, divalent metal-ion-free carboxylase. None of these partial reactions was catalyzed by the small-subunit-depleted, large-subunit octamer to an extent greater than that expected from the residual small subunit content (about 3%), implying that small subunits are required for all three reactions. Clearly, the small subunit's influence is not restricted to any single stage of the catalytic sequence. Under conditions where it was possible to demonstrate tight binding of the reaction-intermediate analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, to the large-subunit octamer, no binding of the 6-carbon intermediate could be detected. We suggest that either the tight-binding form of the 6-carbon intermediate is the hydrated gem-diol, not the ketone, or the large subunits by themselves intrinsically possess a trace of catalytic activity which discharges any bound intermediate before it can be measured.     

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.     

Polypeptide chains of the large and small subunits of fraction I protein from tobacco

Crystalline Fraction I protein from tobacco has been dissociated and separated into three large subunit polypeptides and two small subunit polypeptides by isoelectric focusing in polyacrylamide gels containing 8m urea. The three large subunit polypeptides, resolved by isoelectric focusing, could not be differentiated by amino acid analysis or by fingerprinting of trypsin or chymotrypsin hydrolysates of the individual polypeptides. The two small subunit polypeptides, resolved by hydroxylapatite chromatography in 0.1% sodium dodecyl sulfate as well as by isoelectric focusing, were shown to be distinct polypeptides. The two polypeptides were shown to have different tyrosine:tryptophan ratios, shown by ultraviolet spectra in 0.1m NaOH, and different tyrosine contents shown by amino acid analysis, and they gave different peptide fingerprints after trypsin hydrolysis. The two small subunit polypeptides are concluded to be separate gene products but the three large subunit polypeptides are believed to be the result of modification of a single gene product.     

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.     

Multiple oligonucleotide synthesis in tandem on solid-phase supports for small and large scale synthesis

Multiple oligonucleotides linked end-to-end in tandem can be synthesized by adding a nucleoside to the 5'-OH end of a prior sequence. Nucleosides with 3'-succinyl or Q-Linker arms are coupled with HBTU/DMAP. Alternatively, new phosphoramidite reagents with 3'-ester linkages can be used. Hydroxyl or amino supports can also be used as universal starting materials. Treatment with NH4OH cleaves the 3'-ester to yield only 3'-OH groups and no unwanted 3'-phosphorylated products occur.