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PubMed1992

Kinetics of oxygen binding and subunit assembly for the hemoglobin alpha subunit.

A thorough kinetic characterization of the O2-binding and self-association reactions of alpha-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the alpha O2 self-association reaction. We find an association rate constant of, 2.0 10(5) M(-1)s-1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.

Chemical Phenomena↗
PubMed1970

Three-dimensional organization of the 30S ribosomal proteins from Escherichia coli. I. Preliminary classification of the proteins.

30S ribosomal subunits from Escherichia coli were reacted with three protein reagents. After reaction, the ribosomal proteins were extracted and examined; of the 20 proteins known to compose the particle, nine did not react with any of the three reagents. We have tentatively classified these proteins as "internal," and the remaining eleven as "external." A strong correlation was found between these results and the sequence of assembly. Those proteins which enter the assembly process early are classed as internal, whereas those proteins found to participate later in the assembly sequence are classed as external.

Bacterial Proteins↗
PubMed1985

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli.

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.

DNA Restriction Enzymes↗
PubMed1991

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.

Chromosome Mapping↗
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↗
PubMed1992

In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.

Adenosine Triphosphate↗
PubMed1991

A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli.

Results presented in this study demonstrate that a mutation which inserts an additional tyrosine between the 2 tyrosines at residues 118 and 119 of mature LamB protein results in a temperature-dependent assembly defect. This defect leads to the accumulation of an intermediate at the restrictive temperature that is most likely an assembly-defective monomer. These monomers are rapidly degraded in the wild type (htrA+) strain, and the biphasic kinetics of this degradation indicate that the mutation affects the assembly process and not the final product, i.e. stable trimers. In addition, our data show that the temperature-dependent assembly defect in the mutant strain is reversible, and therefore the accumulated monomers represent a true assembly intermediate. Fractionation studies show that the monomers, which can be accumulated in htrA (degP) mutants at the restrictive temperature, are associated with the outer membrane, indicating that trimerization of LamB is not a prerequisite for localization.

ATP-Binding Cassette Transporters↗
PubMed1991

Phonological assembly in reading: lexical contribution leads to violation of graphophonological rules.

According to current models of reading, the phonological representation of a letter string could be generated by means of two different procedures. The first consists in the looking up or addressing of the phonological representation of the appropriate word in the mental lexicon. The second consists in the assignment of a phonological code to the various orthographic units that occur in the letter string and the assembly of them into a unitary phonological representation. However, the various models of phonological assembly differ in the nature of the knowledge that such assembly requires. On the one hand, dual-route theories assert that the assembly process operates by means of extralexical graphophonological rules and, therefore, without reference to lexical knowledge. On the other hand, in analogy theories and interactive models of phonological assembly, the lexical phonological representations of words orthographically close to the letter string (the orthographic neighborhood) are supposed to contribute to the translation process. The aim of the experiments reported here was to empirically distinguish between these two categories of models. In Experiment 1, subjects were asked to pronounce pseudowords containing the letter g. Results indicate that the phonological representation assigned to the letter g depends on the pronunciation favored by the lexical neighbors. Experiment 2 shows that lexical contribution to phonological assembly also takes place in lexical decision.

Adult↗
PubMed1988

DNA rearrangements affecting both variable and constant regions of Ig H chain genes in MPC11 mouse myeloma variants.

We have examined the mechanisms that account for short Ig H chain production in two variants of the mouse myeloma cell line MPC11 (IgG2b, kappa) by mRNA sequencing and restriction enzyme mapping. One variant, F5.5, has a thymidine residue inserted into the (CH3) domain, of the Ig H chain, resulting in premature termination and translation of a gamma 2b H chain of 50,000 m.w. A second variant, E5.7A12, contains gamma 2a-derived sequences that extend from near the 3' end of the CH2 domain to the intervening sequence between the CH2 and CH3 domains, consistent with a microrecombination event (defined as either a double cross-over or gene conversion event). In this variant, the 5' end of the CH3 domain has been deleted, but the remainder of the gamma 2b(CH3) domain is present, resulting in the translation of a gamma 2b-gamma 2a-gamma 2b H chain of 52,000 m.w. Additional rearrangements affecting sequences in or adjacent to the variable region accompany H chain constant region alterations in these cell lines and subclones of these cell lines. In F5.5, novel sequences have recombined within one of two duplicated copies of the VH gene. In a sister clone of E5.7A12 that has ceased H chain production (E5.7A14), new sequences have recombined within 300 bp 5' of the enhancer element. Both F5.5 and E5.7A12, like their immediate unstable precursor cells, fail to assemble H-H dimers, halting the Ig assembly process at the heavy-light stage, and do not secrete H chains. We speculate that defects in H chain assembly and secretion, as exemplified by the parents of these variants (i.e., intermediates of these secondary variants), reactivate the Ig gene rearrangement machinery and result in the formation of these putatively equally unstable secondary variants.

Amino Acid Sequence↗
PubMed1980

Biosynthesis of two forms of IgM heavy chains by normal mouse B lymphocytes. Membrane and secretory IgM.

A study of the biosynthesis of IgM by purified mouse spleen lymphocytes showed that these cells synthesize both 8 S membrane IgM and 19S secretory IgM, which is identical with plasma cell IgM except in its kinetics of processing, assembly, and secretion. The heavy (mu) chains of these two types of lymphocyte IgM differ in their ultimate fate, in processing, isoelectric point, and peptide composition. The separate precursors of the two mu chains have very similar mobilities in sodium dodecyl sulfate polyacrylamide gel electrophoresis, but they can be distinguished by the use of endoglucosaminidase H (endo-H) to remove core sugars, by two-dimensional electrophoresis, and by one-dimensional gel analysis in pulse-chase experiments. CNBr peptide patterns of intracellular "secretory" mu chains of lymphocytes and plasma cells were similar, but membrane mu chains had a COOH-terminal peptide different in structure from that of secreted mu chains, with a higher apparent molecular weight.

Animals↗
PubMed1981

Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide.

The thermally induced in vitro self-assembly of collagen molecules to form active fibrils illustrates that collagen molecules themselves contain all of the structural information necessary for assembly. The molecule contains three structural domains, the NH2 and carboxyl-terminal extra helical regions (the telopeptides) and the major triple helical rod-like domain. Proteolytic removal of the short telopeptide domains drastically alters the in vitro self-assembly process. We have examined the specific contributions of each telopeptide to the initiation ("nucleation") and growth stages of self-assembly in collagens modified by selective proteinase treatment and by isolating a peptide containing the amino telopeptide and adding this to both normal and proteinase-modified collagen self-assembly systems. Pronase-modified collagen, devoid of both telopeptides, initiated self-assembly very poorly. Addition of small amounts of intact collagen accelerated the rate of nucleation of pronase-modified collagen. Addition of carboxypeptidase-modified collagen also accelerated the nucleation of pronase-modified collagen, suggesting that the remaining amino telopeptide was involved in nucleation. This was confirmed by isolating the cyanogen bromide fragment of the alpha 1(I) subunit containing the amino telopeptide and finding that it specifically accelerated the nucleation of intact pepsin- and pronase-modified to collagens. The amino telopeptide appears to bind to a specific region within the collagen triple helical domain. The isolated peptide requires thermal pretreatment to be active; hence, this interaction must involve a unique telopeptide conformation. This behavior is compatible with the recent model (Helseth, D. L., Jr., Lechner, J. H., and Veis, A. (1979) Biopolymers 18, 3005-3014) proposed for the conformation of the amino telopeptide and its interaction with a helical receptor site as a step in nucleation. Comparison of the behavior of leucine aminopeptidase- and carboxypeptidase-modified collagens suggests that the carboxyl telopeptide has its major role in the growth stages of self-assembly.

Animals↗
PubMed1975

Assembly of the tail of bacteriophage T4.

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a nonaggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sited are limited to growing structures.

Centrifugation, Density Gradient↗
PubMed1987

Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli.

DNA polymerase III holoenzyme was assembled from pure proteins onto a primer template scaffold. The assembly process could be divided into two stages. In the time-consuming first stage, beta subunit and gamma.delta subunit complex were required in forming a tightly bound ATP-activated "preinitiation complex" with a single-stranded DNA bacteriophage circle uniquely primed with a synthetic pentadecadeoxyribonucleotide. This finding substantiates an earlier study using crude protein preparations in a homopolymer system lacking Escherichia coli single-stranded DNA binding protein (Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3511-3515). In the second stage, the polymerase III core and the tau subunit rapidly seek out and bind the preinitiation complex to form DNA polymerase III holoenzyme capable of rapid and entirely processive replication of the circular DNA. ATP is not required beyond formation of the preinitiation complex. It is remarkable that the fully assembled DNA polymerase III holoenzyme is so stably bound to the primed DNA circle (4-min half-time of dissociation), yet upon completing a round of synthesis the polymerase cycles within 10 s to a new preinitiation complex on a challenge primed DNA circle. Efficient polymerase cycling only occurred when challenge primed DNA was endowed with a preinitiation complex implying that cycling is mediated by a polymerase subassembly which dissociates from its accessory proteins and associates with a new preinitiation complex. These subunit dynamics suggest mechanisms for polymerase cycling on the lagging strand of replication forks in a growing chromosome.

Adenosine Triphosphate↗
PubMed1979

Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.

Kinetics↗
PubMed1984

Intracellular site of U1 small nuclear RNA processing and ribonucleoprotein assembly.

We have investigated the intracellular site and posttranscriptional immediacy of U1 small nuclear RNA processing and ribonucleoprotein (RNP) assembly in HeLa cells. After 30 or 45 min of labeling with [3H]uridine, a large amount of U1-related RNA radioactivity in the cytoplasm was found by using either hypotonic or isotonic homogenization buffers. The pulse-labeled cytoplasmic U1 RNA was resolved as a ladder of closely spaced bands running just behind mature-size U1 (165 nucleotides) on RNA sequencing gels, corresponding to a series of molecules between one and at least eight nucleotides longer than mature U1. They were further identified as U1 RNA sequences by gel blot hybridization with cloned U1 DNA. The ladder of cytoplasmic U1 RNA bands reacted with both RNP and Sm autoimmune sera and with a monoclonal Sm antibody, indicating a cytoplasmic assembly of these U1 RNA-related molecules into complexes containing the same antigens as nuclear U1 RNP particles. The cytoplasmic molecules behave as precursors to mature nuclear U1 RNA in both pulse-chase and continuous labeling experiments. While not excluding earlier or subsequent nuclear stages, these results suggest that the cytoplasm is a site of significant U1 RNA processing and RNP assembly. This raises the possibility that nuclear-transcribed eucaryotic RNAs are always processed in the cell compartment other than that in which they ultimately function, which suggests a set of precise signals regulating RNA and ribonucleoprotein traffic between nucleus and cytoplasm.

Autoantibodies↗
PubMed1989

Structure and biological activity of basement membrane proteins.

Collagen type IV, laminin, heparan sulfate proteoglycans, nidogen (entactin) and BM-40 (osteonectin, SPARC) represent major structural proteins of basement membranes. They are well-characterized in their domain structures, amino acid sequences and potentials for molecular interactions. Such interactions include self-assembly processes and heterotypic binding between individual constituents, as well as binding of calcium (laminin, BM-40) and are likely to be used for basement membrane assembly. Laminin, collagen IV and nidogen also possess several cell-binding sites which interact with distinct cellular receptors. Some evidence exists that those interactions are involved in the control of cell behaviour. These observations have provided a more defined understanding of basement membrane function and the definition of new research goals in the future.

Animals↗
PubMed1990

A role for the aspartyl protease from the human immunodeficiency virus type 1 (HIV-1) in the orchestration of virus assembly.

Functional HIV-1 protease (PR) is required for the maturation of viral proteins, for the appearance of characteristic structural features in the virion (as determined by electron microscopy), and for the final assembly of mature virus. Most importantly, HIV-1 PR activity is required for the development of infectivity. Still largely undefined, however, is the timing and control of protease action in this assembly process. Based on the three-dimensional structure of HIV-1 PR2,3 and experimental data reported in the literature, we propose a comprehensive virus assembly model that highlights the role of HIV-1 PR, suggests further experiments to verify the validity of the model, and poses specific questions relevant to the ultimate exploitation of HIV-1 protease as a therapeutic target.

Animals↗
PubMed1988

Studies on DNA topoisomerase activity during in vitro chromatin assembly.

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type II Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it does diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase II may promote extended chromatin assembly.

Animals↗