Formation Of Subunits Research Articles

Induction of microtubule catastrophe by formation of tubulin-GDP and apotubulin subunits at microtubule ends.

The recent discovery that GTP linked to latex beads binds to microtubule ends suggested that nucleotide interactions at this site may play a role in regulating microtubule (MT) dynamics. Evidence for this was sought using DIC microscopy to analyze effects of the free GTP and GDP concentration on the rates of MT elongation and phase transition to rapid shortening (catastrophe, kc). That nucleotide can dissociate and thereby destabilize the plus end by forming nucleotide-free (apotubulin) subunits was indicated by an increase in kc from 0.001 to 0.05 s-1, when the free GTP concentration was reduced from 100 to 0.5 microM, during assembly with 15 microM tubulin--GTP subunits (TuT). That nucleotide can bind to the minus end was indicated by a nearly 5-fold decrease in the rate of elongation when the free GDP concentration was increased from 1.6 to 175 microM, during assembly with a mixture of 36 microM TuT and 54 microM TuD. Further evidence that nucleotide can bind to both ends was provided by the observation that with a mixture of 36 microM TuT and 54 microM TuD, kc was increased from 0.0036 to 0.05 s-1 at the plus end, and from 0.0005 to 0.005 s-1 at the minus end, when the free GDP concentration was increased from 1.6 to 175 microM. Our evidence for destabilization of microtubules by formation of apotubulin and by nucleotide exchange to form terminal TuD subunits suggests that microtubule dynamics can be regulated in cells by an exchange factor that generates apotubulin subunits, or by a GTPase activating protein that forms TuD subunits at microtubule ends.

Ethanol Stimulates Expression of Functional H+,K+-ATPase in SF9 Cells

The baculovirus expression system is suitable for functional expression of gastric H+,K+-ATPase. Expression of functional H+,K+-ATPase in SF9 cells is accompanied by synthesis of large amounts of non-functional subunits. When H+,K+-ATPase is synthesised in the presence of 150-250 mM ethanol in the culture medium, two to threefold higher levels of functional H+,K+-ATPase are produced due to the formation of more functional subunits rather than to an increase of subunits per se. The catalytical properties of the ethanol-produced H+,K+-ATPase are indistinguishable from control preparations. The mechanism by which ethanol stimulates the formation of functional H+,K+-ATPase probably involves a direct effect on the physical properties of Sf9 membranes. In addition there also might be an indirect effect through ethanol inducible stress proteins acting as molecular chaperones.

Transcriptional activation through the tetrameric complex formation of E4TF1 subunits.

Transcription factor E4TF1 is composed of two types of subunit, an ets-related DNA binding protein, E4TF1-60, and its associated proteins with four tandemly repeated Notch-ankyrin motifs, E4TF1-53 and E4TF1-47. To determine the functional domains, we constructed various mutants of the subunits. E4TF1-60 bound to DNA as a monomer. The ets domain and its N-terminal flanking region were necessary to recognize the specific DNA sequence. The 48 amino acids at the E4TF1-60 C-terminus were required for interaction with the other type of subunit. E4TF1-53 and E4TF1-47 share the N-terminal 332 amino acids but differ at the C-termini. They interacted with E4TF1-60 through the N-terminal flanking region to form a heterodimer. E4TF1-53 dimerized with itself, whereas E4TF1-47 did not. The C-terminal region specific for E4TF1-53 was required for the dimerization. Therefore, heterodimers composed of E4TF1-53 and E4TF1-60 were further dimerized, resulting in the formation of a tetrameric complex, which stimulated transcription in vitro. Heterodimers of E4TF1-47 and E4TF1-60 weakly stimulated transcription in vitro. The results indicated that the tetrameric complex formation of E4TF1 subunits was necessary to activate transcription efficiently in vitro.

Functional Requirement of a Site-Specific Ribose Methylation in Ribosomal RNA

The product of the PET56 nuclear gene of Saccharomyces cerevisiae was shown to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S rRNA). Cells reduced in this activity were deficient in formation of functional large subunits of the mitochondrial ribosome. The purified Pet56 protein catalyzed the site-specific formation of 2'-O-methylguanosine on in vitro transcripts of both mitochondrial 21S rRNA and Escherichia coli 23S rRNA. These results provide evidence for an essential modified nucleotide in rRNA.

Coupling of rRNA Transcription and Ribosomal Assembly in Vivo: Formation of Active Ribosomal Subunits in Escherichia coli Requires Transcription of rRNA Genes by Host RNA Polymerase which Cannot be Replaced by Bacteriophage T7 RNA Polymerase

Transcription of a plasmid-located rrnB operon and the corresponding formation of ribosomes in vivo were studied using either T7 RNA polymerase or host RNA polymerase as transcriptase. The 23 S rRNA gene on the plasmid carried an A1067 → T mutation, which confers resistance against the drug thiostrepton. The proportion of particles containing plasmid-borne 23 S rRNA versus chromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067. The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes. When the plasmid rrnB operon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded. The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e. particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes. The particles with plasmid-derived rRNAs were also practically inactive in protein synthesis in vitro . However, the rRNA was functional as shown by reconstitution analysis. The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction or rRNA but rather the T7 transcriptase was responsible for the observed effects. However, when the plasmid rrnB operon was transcribed with host RNA polymerse, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro. When the induction of rRNA transcription by T7 RNA polymerase was performed at 25°C instead of 37°C, an almost normal pattern was observed. Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes. Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37°C than the host polymerase. The results suggest that the formation of active ribosomal subunits in vivo requires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assembly in vivo. T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37°C.

Surface topography of phospholipid bilayers and vesicles (liposomes) by scanning tunneling microscopy (STM)

Abstract A technique has been developed to image desiccated and intact, hydrated phospholipid bilayers (liposomes) using scanning tunneling microscopy (STM). This technique involves impregnation of the specimens with osmium tetroxide or uranium acetate to improve conductivity. In desiccated samples, formation of discrete globular subunits of approximately 20 nm diameter was observed, corresponding to the theoretically smallest diameter (and maximum curvature) of unilamellar phospholipid vesicles. Images of structures of 0.7 to 1 nm diameter which correspond to the expected size of phosphate head groups have been resolved in hydrated uranyl acetate-impregnated phospholipid bilayers. This work presents the first successful images of hydrated liposomes by STM. STM is expected to be useful in assessing the molecular organization of liposomes with distinct surface characteristics, molecular processes occurring during membrane dehydration, and the organization of phospholipids in the vicinity of membrane-associated proteins, lectins or polymers.

Complete primary structure of porcine tenascin

Tenascin is an extracellular matrix protein that is postulated to modulate tissue differentiation and cell migration during development. cDNA clones for tenascin were isolated from a cDNA library of adult porcine submaxillary glands. Three forms of tenascin clones were observed which varied with the number (8-10) of fibronectin type III (FN-III) domains. A major form consists of the N-terminal domain involved in the hexamer formation of tenascin subunits, 14 epidermal-growth-factor-like domains, nine FN-III domains, and the fibrinogen-like domain. A minor form with ten FN-III domains has never been described. Another striking feature is the lack of an RGD sequence that has been implicated to be crucial for cell adhesion, whereas RGD is present in both chicken and human tenascin sequences. In the adult, tenascin is expressed in very restricted tissues such as brain and chicken gizzard. A survey of tenascin transcripts in various adult rat normal tissues, including brain, revealed that the transcripts were detected only in submaxillary glands where tenascin expression has never been reported.

Formation of subviral particles by in vitro translation of subgenomic poliovirus RNAs

Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 degrees C and empty capsids at 37 degrees C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.

Influence of cations on the physicochemical and structural properties of aluminosilicate gel precursors. Part 1. Chemical and thermal properties

The influence of the type of alkali cations on the properties of aluminosilicate gels were studied by chemical and thermal analyses of the solid phases precipitated from the batches of the following molar composition: 4.41 Me 2 O:Al 2 O 3 :3 SiO 2 :222.2 H 2 O, where Me + = Na + , Na + + K + , Na + + Li + , and K + . The change in the chemical composition of dry precipitates, influenced by the presence of different cations and their mixtures, is discussed with respect to the interaction of cations with silicate species in the solution and colloid chemistry of gelation processes. Analysis of kinetics of dehydration of gels as well as hydrothermal treatment of the gels confirmed the role of the sodium ion as a “structure-forming” cation and revealed that sodium ions cause the formation of structural subunits inside the gel matrix.

Isolation and partial characterization of a soluble mucin in human pancreatic juice

Morphological and histochemical abnormalities in pancreatic mucin occur in many pancreatic disorders. However, the composition of pancreatic mucin is poorly understood. Purified mucin was isolated from pure pancreatic juice by sequential chromatography on Sepharose CL-2B and CL-4B followed by CsCl density gradient ultracentrifugation. The mucin preparation consists of 24% protein and 73% carbohydrate. Reduction of the macromolecule (greater than 2 x 10(6)) by mercaptoethanol resulted in the formation of subunits of molecular weight 500,000 and released several small molecular weight proteins, including a glycoprotein of an average molecular weight of 116,000. Cellulose acetate electrophoresis separated the mucin into three species of different staining properties for periodic acid-Schiff reagent and Alcian blue, suggesting the presence of microheterogeneity with respect to sulphation and sialation. Threonine, serine, and proline composed 48% of the total amino acids, while the oligosaccharide moiety contained N-acetylglucosamine, N-acetylgalactosamine, fucose, galactose, sialic acid, and sulphate. We also detected the presence of C16:0 and C18:0 fatty acids which were probably noncovalently bound to the pancreatic mucin.

A stochastic branching model with formation of subunits applied to the growth of intestinal crypts

The intestinal epithelium is one of the most rapidly regenerating tissues in mammals. Cell production takes place in the intestinal crypts which contain about 250 cells. Only a minority of 1-60 proliferating cells are able to maintain a crypt over a long period of time. However, so far attempts to identify these stem cells were unsuccessful. Therefore, little is known about their cellular growth and selfmaintenance properties. On the other hand, the crypts appear to exhibit a life cycle which starts by fission of existing crypts and ends by fission or extinction. Data on these processes have recently become available. Here, we demonstrate how these data on the life cycle of the macroscopic crypt structure can be used to derive a quantitative model of the microscopic process of stem cell growth. The model assumptions are: (1) stem cells undergo a time independent supracritical Markovian branching process (Galton-Watson process); (2) a crypt divides if the number of stem cells exceeds a given threshold and the stem cells are distributed to both daughter crypts according to binomial statistics; (3) the size of the crypt is proportional to the stem cell number. This model combining two different stochastic branching processes describes a new class of processes whose stationary stability and asymptotic behavior are examined. This model should be applicable to various growth processes with formation of subunits (e.g. population growth with formation of colonies in biology, ecology and sociology). Comparison with crypt data shows that intestinal stem cells have a probability of over 0.8 of dividing asymmetrically and that the threshold number should be 8 or larger.

The conserved GTPase center and variable region V9 from Saccharomyces cerevisiae 26S rRNA can be replaced by their equivalents from other prokaryotes or eukaryotes without detectable loss of ribosomal function.

Using the "tagged" rRNA gene system, which allows in vivo mutational analysis of Saccharomyces cerevisiae rRNA, we studied the role of two distinct structural elements of 26S rRNA in ribosome biogenesis and function--namely, the evolutionarily highly conserved "GTPase center" located in domain II and the eukaroyote-specific variable region V9 in domain III. Replacement of the S. cerevisiae GTPase center with its counterpart from Escherichia coli did not affect the assembly of the mutant 26S rRNA into functional (as judged by their polysomal distribution) 60S subunits, indicating that the E. coli GTPase center functions efficiently in the context of the heterologous rRNA. Removal of most of the S. cerevisiae V9 region or replacement of this segment by the equivalent segment from mouse 28S rRNA also did not affect the formation of functional 60S subunits carrying the mutant 26S rRNA. Therefore, the V9 region does not seem to play a role in the biological functioning of the yeast 60S subunits, and these subunits appear to be able to accommodate V9 regions of various size and secondary structure without apparent loss of function.

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different α-major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1α, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.

Functional analysis of transcribed spacers of yeast ribosomal DNA.

Making use of an rDNA unit, containing oligonucleotide tags in both the 17S and 26S rRNA gene, we have analyzed the effect of various deletions in the External Transcribed Spacer (ETS) and in one of the Internal Transcribed Spacers 1 (ITS1) on the process of ribosome formation in yeast. By following the fate of the tagged transcripts of this rDNA unit in vivo by Northern hybridization we found that deleting various parts of the ETS prevents the accumulation of tagged 17S rRNA and its assembly into 40S subunits, but not the formation of 60S subunits. Deleting the central region of ITS1, including a processing site that is used in an early stage of the maturation process, was also found to prevent the accumulation of functional 49 S subunits, whereas no effect on the formation of 60S subunits was detected. The implications of these findings for yeast pre-rRNA processing are discussed.

Neural interactions mediating the detection of motion in the retina of the tiger salamander

The neural circuitry underlying movement detection was inferred from studies of amacrine cells under whole-cell patch clamp in retinal slices. Cells were identified by Lucifer yellow staining. Synaptic inputs were driven by "puffing" transmitter substances at the dendrites of presynaptic cells. Spatial sensitivity profiles for amacrine cells were measured by puffing transmitter substances along the lateral spread of their processes. Synaptic pathways were separated and identified with appropriate pre- and postsynaptic pharmacological blocking agents. Two distinct amacrine cell types were found: one with narrow spread of processes that received sustained excitatory synaptic current, the other with very wide spread of processes that received transient excitatory synaptic currents. The transient currents found only in the wide-field amacrine cell were formed presynaptically at GABAB receptors. They could be blocked with baclofen, a GABAB agonist, and their time course was extended by AVA, a GABAB antagonist. Baclofen and AVA had no direct affect upon the wide-field amacrine cell, but picrotoxin blocked a separate, direct GABA input to this cell. The narrow-field amacrine cell was shown to be GABAergic by counterstaining with anti-GABA antiserum after it was filled with Lucifer yellow. Its narrow, spatial profile and sustained synaptic input are properties that closely match those of the GABAergic antagonistic signal that forms transient activity (described above), suggesting that the narrow-field amacrine cell itself is the source of the GABAergic interaction mediating transient activity in the inner plexiform layer (IPL). Other work has shown a GABAB sensitivity at some bipolar terminals, suggesting a population of bipolars as the probable site of interaction mediating transient action. The results suggest that two local populations of amacrine cell types (sustained and transient) interact with the two populations of bipolar cell types (transient forming and nontransient forming). These interactions underlie the formation of the change-detecting subunits. We suggest that local populations of these subunits converge to form the receptive fields of movement-detecting ganglion cells.

ChemInform Abstract: Mechanism of the Formation of the Two Epimeric Diamino‐hexose Rings of Neomycin B.

AbstractBy using variously tritiated hexoses [(6RS)‐D‐[1‐14C,6‐T]glucosamine,(6RS)‐D‐[6‐14C,6‐T]glucose, D‐[U‐14C,5‐T]glucose, D‐[U‐14C′3,4‐T2]glucose] and Streptomyces fradiae (a neomycin producer), the mechanism and stereochemistry of the formation of various subunits of neomycin B (I) are investigated.

Peptic hydrolysis of gluten, glutenin and gliadin from wheat grain: Kinetics and characterisation of peptides

AbstractThe kinetics of the hydrolysis of gluten, glutenin and gliadin by pepsin was studied by following the reaction of the hydrolysate with ninhydrin and by SDS‐gel electro‐phoresis. Glutenin was more rapidly hydrolysed than native molecules of gliadin and produced components of different size. The first stage of hydrolysis of gliadin resulted in the formation of fragments mainly in the range 25 000 and 10 000 daltons whereas polypeptides generated from glutenin had subunits of molecular weight (MW) in the range 65 000, 30 000 and 10 000. Further hydrolysis resulted in the formation of subunits of MW around 24 000 and 14 000 for glutenin and gliadin respectively. The final stage of hydrolysis produced a quantity of peptides of MW under 10 000 and a small quantity of free amino acids. No obvious differences in the molecular weights of the polypeptides released were found when the enzyme to gliadin ratio was varied. Components obtained from glutenin and gliadin were further characterised after gel filtration on Sephadex G50. Large polypeptides derived from both gliadin and glutenin contained large amounts of proline and glutamate. Surface hydrophobicity of derived components were different, and inferior to, those of the reduced glutenin and native gliadin. Reduction of disulphide bonds of digested polypeptides of glutenin showed the existence of peptides branched by interchain disulphide bonds. Peptic cleavage sites were probably located differently in glutenin and gliadin molecules.

Mechanism of the formation of the two epimeric diamino-hexose rings of neomycin B

By using variously tritiated hexoses and Streptomyces fradiae(a neomycin-producer), the mechanism and stereochemistry of the formation of various subunits of neomycin B were investigated. The results of the incorporation of (6RS)-D-[6-3H] glucose into neomycin B followed by specific degradation showed that during the formation of the aminomethyl group of the neosamine B ring one of the 6-3H atoms of the precursor was removed. The 3H remaining at C-6 of the neosamine B ring was shown to be located in the Hsi orientation through the isolation of the chiral centre as glycine and the analysis of the latter by using an exchange reaction catalysed by serine hydroxymethyltransferase. These results suggested that, as has been previously shown for neosamine C, the aminomethyl group of neosamine B is formed by an oxidation-transamination process; –CH2OH →–CHO →–CH2NH2 D-[5-3H]Glucose was prepared by an unambiguous method and incorporated into neomycin B. The radiochemical data on the various subunits of the antibiotic could be interpreted to suggest that the L-idose configuration at C-5 of the neosamine B ring is produced via an enolisation process involving a carbonyl group at either C-4 or C-6. The involvement of C-4 was then eliminated by feeding experiments in which it was shown that the hydrogen atom at C-4 of D-glucose was undisturbed during the incorporation of this position into the C-4 of neosamine B or C.

Dynamic model of gonadotropin-releasing hormone receptor function.

Functioning gonadotropin-releasing hormone (GnRH) receptor is visualized as an aggregate of identical subunits (not all always functional) with the aggregate usually transformed into at least four successive structurally distinct receptor assemblies. Receptor protein, hormone molecule(s), and carrier(s) are main components of each functional subunit. During the normal lifespan of the functional subunit, each carrier is responsible for delivery of a unit amount of product, per unit time, to the cell surface (ratio between functional carrier and bound hormone, 1:1). Association of hormone with the receptor protein is essential, not only for the initial formation of the functional subunits but also for subsequent conformational changes that are in turn essential for formation of the aggregate only, or later (in the presence of sufficiently high GnRH concentrations) for a successive formation of a family of receptor assemblies (occurring one at a time). The successive assemblies differ from the aggregate by being more stable and from one another by increasing GnRH binding affinity and apparent capacity. They resist stimulation during protracted decay (desensitization).

Hydrophobic binding properties of bovine gallbladder mucin.

Hydrophobic binding properties of purified bovine gallbladder mucin were studied by fluorescence spectroscopy using 1-anilino-8-naphthalene sulfonate (ANS) and N-phenyl-1-naphthylamine. The purified glycoprotein contained 75.5%, dry weight, as carbohydrate, 16.3% as protein, and 3.7% as sulfate; Mr = 2.2 X 10(6) was estimated by chromatography on Sephacryl S-500. Mucin contained a large number of low-affinity binding sites for these hydrophobic ligands. The dissociation constant, KD of mucin-ANS binding was 2.7 X 10(-5); each mucin molecule had approximately 42 binding sites for ANS. These binding sites were deduced to be on the unglycosylated portion of the protein core, as Pronase digestion completely eliminated binding. Reduction of mucin with 2-mercaptoethanol increased the fluorescence yield by formation of subunits with increased binding sites for the ligand. Increasing NaCl concentration (0.125 to 2.0 M) and decreasing pH (9 to 3) progressively increased fluorescence with the charged ligand ANS, suggesting that the binding site may have acidic groups which are shielded at high ionic strength or low pH. The fluorescent yield with N-phenyl-1-naphthylamine, an uncharged ligand, was an order of magnitude higher than with ANS. Bilirubin and bromosulfophthalein inhibited mucin-induced ANS fluorescence, but bile acids did not. Gallbladder mucin contains hydrophobic binding domains in the nonglycosylated peptide core that are involved in polymer formation and binding of biliary lipids and pigment.