Insoluble Polypeptides Research Articles

Effect of Dry Heat Temperature and Moist Heat Pressure on Canola Meal for Ruminant Utilisation. Part II: Maillard Reaction Product Formation and Structural Characteristics

The effects of a range of barrel dry heat temperatures (20 to 180 oC), and moist heat pressure (MHP) (120 oC 15 min 192 kPa) on Maillard reaction product (MRP) formation and canola meal structural characteristics were investigated. Increasing dry heat temperature was negatively correlated with meal whiteness L* and yellowness b* (early-MRP) and positively with surface hydrophobicity. Relative to control meal, MHP increased early-MRP, redness, browning index (late-MRP), and acidity; and decreased L*, surface hydrophobicity, b*, and Abs294nm (intermediate-MRP). Dry heat-associated changes in surface hydrophobicity suggest protein unfolding and side-chain modifications. Lack of high MW polypeptides at dry heat temperatures of 160 and 180 oC imply protein denaturation and formation of insoluble polypeptides. Specific dry heat temperatures increased surface lipid and induced the formation of protein matrix and aggregation. Meal surface morphology rounded and flattened at specific dry heat temperatures, and smoothed with MHP. Differences in lipid-related functional groups were evident between dry heat temperatures, and with MHP. Treatment with MHP affected amide I and II, α-helix, β-sheet, their respective ratios and the total protein fingerprint region; fragmented meal into proteolysis-resistant protein aggregates with crevices containing lipid droplets; and, reduced solubility of canola meal polypeptides > 40 kDa. The changes observed may have a great effect on ruminal degradation and supply of protein and AA for ruminant utilisation.

Globulin Expression in Grain of Australian Hard Wheat Cultivars Is Affected by Growth Environment

ABSTRACTOur aim was to study changes in wheat proteomes across different growth locations as the first step in linking protein composition with functional changes in grains produced with commercial production systems. Soluble and insoluble proteins were extracted sequentially from grain of three commercial wheat cultivars grown at four locations in New South Wales, Australia, during a single season. Bands were separated with SDS‐PAGE and identified by peptide mass fingerprinting. Quantitative changes in the electrophoretic patterns were observed mainly in the insoluble polypeptides of molecular mass 40,000–70,000 for all three cultivars grown at two of the four locations. These proteins were identified as mainly globulin and serpin isoforms, as well as triticin. Other proteins with changed expression included disease‐resistance proteins, class III peroxidase, starch branching enzyme I, β‐amylase, and storage proteins. Two‐dimensional electrophoretic analysis was performed on two of the same wheat cultivars grown at one of the locations during two consecutive seasons. Protein spots that varied between seasons consisted of globulin and serpin isoforms, triticin, HMW glutenin, γ‐gliadin, starch branching enzyme IIb, and α‐amylase. The implications of the upregulation of globulin and triticin on whole meal flour quality, through their participation in polymerization of the gluten network, are considered.

Chemoenzymatic Synthesis of Poly(l-alanine) in Aqueous Environment

L-alanine ethyl ester was polymerized into poly(L-alanine) (polyAla), one of the insoluble polypeptides, by papain in aqueous buffer at varying pH. At neutral pH, a maximum chain length of 11 repeats was observed. These polymers were dominated by random coiled structure and demonstrated a lack of patterned macromolecular assembly. Under alkaline conditions, longer polymer chain lengths were achieved, and the maximum chain length was 16 repeats. These longer chains showed distinct β-sheet formation and were capable of fibril assembly. The present study reports on chemoenzymatic synthesis of a hydrophobic homopolypeptide under aqueous conditions as well as demonstrates a chain length dependency of secondary structure formation and macromolecular assembly of chemoenzymatically synthesized polyAla, providing a new insight into material design of polypeptide.

Nociceptive reaction-related state of glial intermediate filaments in the brain of rats with hyperfunction of the thyroid gland: An ontogenetic aspect

In experiments on Wistar rats of two age groups (5 weeks old and mature, 5 months old), we studied the content and polypeptide composition of glial fibrillary acidic protein (GFAP) in the cerebral cortex, hippocampus, thalamus, and brainstem under conditions of experimentally induced hyperfunction of the thyroid gland, as well as of combination of hyperthyroidism with the pain model (consequences of laparotomy). The hyperthyroid state was created by administration of L-thyroxine (initial dose, 10 µg/day) with food for 2 weeks; the dose was increased daily by 10 µg. At the end of experiment, we measured the level of thyroxine in the blood serum using an immunoenzymatic technique. We found that in the hyperthyroid state 5-week-old rats showed a significant increase in the content of filamentous GFAP fraction in the hippocampus, while when this influence was combined with the postoperation pain syndrome, an increase was observed in the brainstem (by 18 to 27%). Results of immunoblotting demonstrated that degraded polypeptides with a molecular mass of 47 to 45 kdalton were manifested under these conditions, which is indicative of intensification of cytoskeletal rearrangement of astroglia. In mature rats under the same conditions, we observed a drop in the level of insoluble polypeptides of intermediate filaments in astrocytes in the thalamus, hippocampus, and cerebral cortex.

The Acrolepiopsis assectella silk cocoon: kairomonal function and chemical characterisation

Two soluble sericin-like polypeptides, B1 and B2, from leek moth ( Acrolepiopsis assectella) cocoons trigger host-acceptance behaviour in the parasitoid, Diadromus pulchellus (Proc. Roy. Soc. London B 269 (2002) 1879). We found that these polypeptides were particularly cysteine-rich and lost their ability to trigger host-acceptance behaviour after being denatured and purified. This suggests that inter-disulphide bonds and the secondary structure of B1 and B2 are important for their biological activity. We also isolated six insoluble polypeptides (or polypeptides of low solubility) from A. assectella cocoons. At least four of these polypeptides triggered host-acceptance behaviour. The strongest responses were observed with P22, a light-chain fibroin or a seroin-peptide, and P100, a sericin-like polypeptide that is probably more strongly associated with the silk core than are B1 and B2. In conclusion, several polypeptides from different parts of the A. assectella silk-cocoon (the insoluble core and coating of the silk thread) are able to elicit host-acceptance behaviour in D. pulchellus females. These polypeptides belong to different silk protein families and are used as kairomones by this specialist parasitoid.

Biochemical Assay Detects Feeding Damage to Loblolly Pine Seeds Caused by the Leaffooted Pine Seed Bug (Hemiptera: Coreidae)

A large number of proteins in salivary gland extracts of the leaffooted pine seed bug, Leptoglossus corculus Say, were strongly recognized by a polyclonal antibody-based assay developed for detecting saliva of the western conifer seed bug. Leptoglossus occidentalis Heidemann, in lodgepole pine, Pinus contorta var. latifolia Engelmann, seeds. An average of approximately 85% of loblolly pine, Pinus taeda L., seeds exposed to feeding by L. corculus for 1 to 4 weeks in the laboratory contained detectable amounts of salivary proteins when the antibody assays were performed weekly on samples (n = 10) of seed. In comparison, radiography of exposed seed detected an average of approximately 63% damaged seed over the same 4-wk period, indicating that the antibody assay increased sensitivity of damage detection by approximately one-third. Depletion of insoluble polypeptides and proliferation of soluble polypeptides ≤23.5 kDa was apparent after SDS-PAGE and quantitative assays were performed on proteins extracted from seeds that were damaged by exposure to L. corculus feeding. Our data suggest that the antibody-based test could be used to obtain accurate estimates of seed losses attributable to L. corculus feeding in southern pine seed orchards.

The cDNA Sequences of Three Tetrins, the Structural Proteins of the Tetrahymena Oral Filaments, Show that they are Novel Cytoskeletal Proteins

The oral filaments of the ciliate Tetrahymena consist of the tetrins, insoluble polypeptides with molecular masses of around 85 kD. We characterised the tetrins of T. thermophila by two-dimensional gels and derived a large number of peptide sequences by in gel digestion. Using RT-PCR techniques and RACE-PCR, the complete cDNA sequences of tetrins A, B and C were established. Although tetrins differ strikingly in protein sequence they show a common structural principle. A N-terminal domain of 60 to 100 residues contains most of the proline residues of the tetrins and is probably globular. It is followed by a long alpha-helical domain of 620 to 640 residues which either lacks prolines or in tetrin A contains a single proline residue. Although this long domain has coiled coil forming ability, the individual heptad repeats are not extensive. Tetrins are novel cytoskeletal proteins unique to ciliates. Since the three tetrin sequences account for all 900 amino acid residues obtained by microsequencing of peptides, an additional major tetrin seems excluded. A minor component D is related to tetrin B by peptide sequences. The isoelectric variants, particularly obvious for tetrin A, most likely reflect post-translational modifications. These could arise by phosphorylation of serines and threonines in the proline rich N-terminal domain.

Effects of Dissociation, Deamidation, and Reducing Treatment on Structural and Surface Active Properties of Soy Glycinin

Different soluble protein derivatives were prepared from soy glycinin by means of progressive dissociation, deamidation, and later reducing thermal treatment. Dissociation was achieved at 4 °C in low ionic strength or mild acid conditions (0.1 M HCl). For deamidation, mild acid treatment of glycinin was performed at 70 °C for 1.5-9 h. Deamidation levels (1.7-25.0%) increased linearly with time of heating. These results were compared to those obtained by alkaline deamidation. The reducing thermal treatment led to both soluble and insoluble polypeptides. Greater soluble protein yields were obtained when this treatment was applied to previously deamidated glycinins. The structural properties and surface activity of native and modified glycinin were studied. Dissociation, deamidation, and reduction resulted in structural changes on glycinin (increased surface hydrophobicity, decreased molecular size, and isoelectric pH) which enhanced adsorption of proteins at the air-water interface. In contrast to native glycinin, the influence of ionic strength on surface behaviors is rather limited when the protein is both deamidated and reduced.

Overexpression of aRhizopus delemar lipase gene inEscherichia coli

A cloned complementary deoxyribonucleic acid encoding the precursor polypeptide of an extracellular lipase from the fungus Rhizopus delemar was altered by site-directed mutagenesis to generate deoxyribonucleic acid fragments that specifically code for the polypeptides of the proenzyme and the mature form of the lipase. Attempts to produce these polypeptides in enzymatically active form in Escherichia coli revealed toxic effects toward the host. Therefore the polypeptides were expressed as inactive and insoluble forms in the cytoplasm of E. coli BL21 (DE3) cells using plasmid vector pET11-d. With this tightly regulated high-level expression system, lipase and prolipase polypeptides were produced to estimated levels of up to 21% and 15%, respectively, of total cellular protein. The insoluble polypeptides were solubilized in 8 M urea. Refolding into active forms was achieved by treatment with the redox system cystine/cysteine and dilution. Refolded mature lipase was purified to homogeneity by affinity and ion exchange chromatography. The enzyme had a specific activity comparable to that of lipase from the fungal culture. The quantities of pure enzyme obtained from a 1-L culture of E. coli exceeded those obtained from the fungal culture by a factor of at least 100. Refolded recombinant prolipase was purified essentially to homogeneity and had a specific activity similar to that of the mature enzyme. Its pH optimum was 7.5, rather than the pH 8 determined for recombinant mature lipase and for the enzyme purified from the fungal culture. Recombinant prolipase retained activity after 15 min incubation at 65 degrees C, while mature lipase retained activity only up to 45 degrees C.

Differences of pancreatic stone morphology and content in patients with pancreatic lithiasis

Pancreatic stones from 25 patients were compared by morphological and/or radiological examination. Twenty patients, mostly alcoholics, had calcified stones. Five (four nonalcoholic women) had radiolucent stones. Aspect and consistency of calcified stones varied from compact and resistant to coralliform and brittle but were identical in the same patient. In the coralliform type, organic fibrils with a diameter up to 10 microm and a length up to a few centimeters were observed, strongly attached to mineral crystals. The lithostathine (formerly called pancreatic stone protein, PSP) content was estimated in each stone significantly lower in the populations with larger stone mass, compared to populations with small amounts of stones. Transparent stones were built up of an amorphous material solubilized at acidic pH and corresponding to degraded forms of lithostathine-S (S for secretory). In one patient, we followed over seven years the evolution of a radiolucent calculus. We observed that the radiolucent core occurred first, and was secondarily wrapped in a calcified shell. We conclude that morphological differences observed in this study among pancreatic stones suggest that different mechanisms have been involved in their formation. Among them, lithostathine transformation into insoluble polypeptides may provide different types of protein aggregates, some of them being able to promote CaCO(3) apposition and others having no affinity for calcium.

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments.

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton.

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.

Organization and dynamics of a cortical fibrous network of Paramecium: The outer lattice

AbstractA monoclonal antibody, CC212, raised against ciliated cortices of quail oviduct cells and characterized as an antimyosin of smooth muscle and nonmuscle cells, was shown to specifically label a regular cortical network in Paramecium and to recognize two Triton X‐100–insoluble polypeptides at 130 and 50 kDa. However, no evidence was obtained that these polypeptides are related to myosin.An immunofluorescence study and ultrastructural immunogold localization allowed us to identify the CC212‐decorated material as a component of the outer lattice, a submembrane cytoskeletal network which runs along the top of the ridges visible by scanning electron microscopy and delineates the periphery of each cortical unit. The dynamics of the outer lattice during the cell cycle was studied by immunofluorescence and it was found that the outer lattice growth is achieved without disruption of the preexisting meshes by longitudinal elongation and additon of new transverse partitions. A striking disorganization of the outer lattice was observed in a thermosensitive mutant primarily altered in basal body duplication. These observations suggest possible functions of the outer lattice and demonstrate the interdependence of basal body duplication, surface growth, and outer lattice remodelling.

The state of sulphydryl groups in proteins isolated from normal and cataractous human lenses.

Individual crystallins, urea-soluble and urea-insoluble proteins were isolated from the nucleus and cortex of types I-IV cataractous lenses and normal lenses. The levels of protein sulphydryls (P-SH), disulphides (S-S), as well as surface (F-SH) and buried (S-SH) in these proteins were determined by reaction with 5, 5'-dithiotris- (2-nitrobenzoic acid) or performic acid oxidation followed by amino acid analysis. During nuclear colour development there is a progressive decrease in the sulphydryl content of the crystallins. In the nuclei of advanced cataractous lenses, the P-SH decreases to 10% of the levels found in the normal nucleus. Similar but smaller changes take place in the cortex. No specific changes were found between the crystallins, with the exception of beta S crystallin. The cysteine remains constant in all lens types suggesting no higher oxidation products are formed. There is a significant shift in the distribution of cysteine in the nucleus of type III and IV lenses. Urea-insoluble proteins are the predominant species, accounting for about 70% of the total cysteine pool. This is consistent with the accumulation of modified insoluble polypeptides during senile nuclear cataract formation.

Analysis of the nuclear envelope polypeptides by isoelectric focusing and electrophoresis

The more insoluble polypeptides of the avian erythrocyte nuclear envelope have been characterized by a two-dimensional electrophoretic procedure. Most of the polypeptides occur in two classes with isoelectric points of approximately 6.4 and 5.7 respectively. The more acidic class contains two polypeptides, P71 and one which contributes to an electrophoretic band previously identified as P55. The more basic class includes P75, P68, P61 and two or more polypeptides from the P55 band. There are four to six isoelectric point variants of each polypeptide in the more basic class, and the relative stain intensities for the variants are similar for the different polypeptides. These similarities in ionic properties suggest a chemical relationship between the polypeptides. These results are discussed in relation to the in vitro conversion of P75 to polypeptides of the same molecular weight as P68, P61 and P55.

赤外吸収スペクトルによるポリペプチドの分子量の迅速測定法

赤外吸収スペクトルによるポリペプチドの分子量の迅速測定法