Initial kinetics of Protein turnover in growing yeast

Proteins in yeast growing in a medium with glucose or ethanol as carbon source were pulse-labelled by a 20-min incubation with¹⁴C-leucine. The proteins in cells labelled and growing in a glucose medium were stable; when this population was transferred to the ethanol medium, the proteins were degraded at a rate of 1.1 %/h. The population labelled and growing in an ethanol medium displayed a fraction of short-lived proteins (about 4 %), decaying with a half-life of 0.5 h. The size of the short-lived protein fraction increased slightly after shifts to a glucose as well as to a starvation medium. The residual long-lived proteins underwent a turnover of 1.3 –1.4 %/h in the ethanol or the starvation medium and of 0.3 %/h in the glucose medium, respectively. Proteins labelled in the presence of canavanine or ethionine were degraded at only a slightly greater rate than the normal proteins. Participant of the UNESCO Postgraduate Course “On Modern Problems in Biology”.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Attenuation of liver insoluble protein carbonyls: indicator of a longevity determinant?

Oxidative damage affects protein structure and function. Progressive accumulation of oxidized proteins is considered a putative mechanism of aging; however, empirical evidence supporting their role in aging is inconsistent. This inconsistency may reflect a failure to distinguish damage to particular cellular compartments. We found a significant reduction of protein carbonyls in the insoluble, but not in the soluble, fraction of liver tissues of long-lived compared with their short-lived counterpart. Of cellular components analyzed, only nuclear protein carbonyl level was uniformly reduced in long-lived compared with short-lived animals. This observation suggests that attenuated accumulation of protein carbonyls in the nucleus, where they can affect multiple aspects of gene expression and DNA repair, might contribute to the longevity in mammalian species.     

PROTEIN SYNTHESIS IN VITRO IN NUCLEI OF HUMAN NERVOUS TISSUE

Abstract—

• 1 In vitro incorporation of tritiated leucine into nuclear proteins of normal human brain, astrocytoma I and II and glioblastoma multiforme was investigated. The distribution of radioactivity among various protein fractions of nuclei was determined.
• 2 The results demonstrate that the isolated nuclei of astrocytoma I and II incorporate radioactivity into proteins at least 40 times more actively than nuclei of normal human brain or glioblastoma multiforme.
• 3 The residual protein fraction was the most highly labelled fraction among the nuclear pioteins. This fraction from astrocytomas incorporates relatively more radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The buffered-saline soluble protein fraction of astrocytoma nuclei contained a relatively lower amount of radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The total radioactivity incorporated into the deoxyribonucleoproteins seemed to increase with the malignancy of the tissue investigated. The significance of the results with respect to malignant transformation is discussed.

     

SProtP: a web server to recognize those short-lived proteins based on sequence-derived features in human cells

Protein turnover metabolism plays important roles in cell cycle progression, signal transduction, and differentiation. Those proteins with short half-lives are involved in various regulatory processes. To better understand the regulation of cell process, it is important to study the key sequence-derived factors affecting short-lived protein degradation. Until now, most of protein half-lives are still unknown due to the difficulties of traditional experimental methods in measuring protein half-lives in human cells. To investigate the molecular determinants that affect short-lived proteins, a computational method was proposed in this work to recognize short-lived proteins based on sequence-derived features in human cells. In this study, we have systematically analyzed many features that perhaps correlated with short-lived protein degradation. It is found that a large fraction of proteins with signal peptides and transmembrane regions in human cells are of short half-lives. We have constructed an SVM-based classifier to recognize short-lived proteins, due to the fact that short-lived proteins play pivotal roles in the control of various cellular processes. By employing the SVM model on human dataset, we achieved 80.8% average sensitivity and 79.8% average specificity, respectively, on ten testing dataset (TE1-TE10). We also obtained 89.9%, 99% and 83.9% of average accuracy on an independent validation datasets iTE1, iTE2 and iTE3 respectively. The approach proposed in this paper provides a valuable alternative for recognizing the short-lived proteins in human cells, and is more accurate than the traditional N-end rule. Furthermore, the web server SProtP (http://reprod.njmu.edu.cn/sprotp) has been developed and is freely available for users.     

NUCLEAR LOCALIZATION OF S-100 PROTEIN

Abstract— S-100 protein has been found in the nuclei isolated from the brain cortex of rabbit. The nuclear S-100 constitutes a small portion (0.55 per cent) of the S-100 present in the cytosol. Most of the large and pale nuclei appear to contain much more S-100 than the small and dark ones. The nuclear membrane is permeable in vitro to S-100 in presence of divalent cations. Three forms of S-100 occur in subnuclear fractions: free S-100, present in the soluble protein fraction; labile-bound S-100, present in the deoxyribonucleoprotein fraction and stable-bound S-100, present in the residual or‘nucleolar’fraction. The localization of the S-100 in those regions of the nucleus that are most active in RNA synthesis provides basic information for further studies on the possible role of this protein on genomic expression in nervous tissue.     

Distribution,Speciation and Bioaccumulation of Hg and As in Mariculture Sediments from Dongshan Bay,China

Hg and As are the major hazardous pollutants in marine sediments due to their high toxicity to benthonic organisms. Understanding the spatial distribution, speciation and bioaccumulation of these toxic elements in sediments is therefore of high environmental importance for identifying their potential risks. Sediments and bivalves Paphia undulata were collected from the mariculture area of Dongshan Bay, China, for characterizing geochemistry (by using the European Community Bureau of Reference (BCR) sequential extraction procedure) and bioaccumulation of Hg and As [by calculating the biota sediment accumulation factor (BSAF)]. Both elements in sediments were mostly associated with the residual fraction (69.52–95.06% and 88.22–91.12% of the total concentration, respectively), followed by the oxidizable (bound to sulfides and organic matter) fraction (1.25–25.32% and 3.62–6.00%, respectively). However, Hg presented a higher bioaccumulation than As. Correlation analysis indicated that As in residual fraction and Hg in oxidizable fraction exert positive contributions (R = 0.927, P < 0.01 and R = 0.869, P < 0.05, respectively) on their own bioaccumulation factor. This indicated that P. undulata could adsorb both Hg in organic fraction and As in residual fraction from the sediments. Therefore, we should pay more attention to the potential dissolution and release of metals bound to sediments in the digestive tracts of marine organisms.     

Selenium distribution in ryegrass and its antioxidant role as affected by sulfur fertilization

Selenium (Se) essentiality to plants has not been demonstrated although evidence indicates that it plays a significant role as antioxidant in higher plants. Research concerning to the uptake and allocation of Se in plant tissues is reported in numerous works. However, the effect of sulfur (S) on both the distribution and the antioxidant ability of Se in selenite-treated plant remains unclear. In this work the effect of S application (0–100 mg S kg⁻¹ soil) on shoot Se concentration of Lolium perenne cv. Aries was studied. Se distribution into different fractions of plants supplied with selenite (2 mg Se kg⁻¹ soil) and the state of the antioxidative system were determined. Results showed that shoot Se concentration decreased at least 33% by S application. Plants supplied with S registered the lowest GSH-Px activity and the highest lipid peroxidation. Most of Se was incorporated into the organic fraction of the plant tissue irrespective of the S treatment. However, a significant decrease of both the soluble protein and the amino acid fraction occurred, and the residual Se fraction seemed to increase at expense of the organic-Se soluble fraction. Although no essential selenoproteins have been clearly identified in vascular plants, the decrease of the soluble protein fraction and the different pattern of protein synthesized (SDS-PAGE analysis) may explain the observed reduction of the GSH-Px activity.     

Import of rat liver mitochondrial transhydrogenase.

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.     

Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Affinity purification of a 65-kilodalton parasporal protein from Bacillus thuringiensis PG-14 that shows mosquitocidal activity

By using antibody-mediated affinity chromatography, a highly mosquito larvicidal but nonhemolytic fraction was obtained from alkali-solubilized, silkworm (Bombyx mori) larval gut juice-treated parasporal inclusions of Bacillus thuringiensis strain PG-14 (serotype 8a : 8b). This fraction contained a 65-kDa protein only but not a 25-kDa protein, the main component in the flow through fraction unbound to the affinity column. The 25-kDa protein purified from the unbound fraction by CM-cellulose chromatography demonstrated a high hemolytic activity against sheep red blood cells but very low mosquito larvicidal activity.     

Studies on the incorporation of labelled sulphate into cells and cell-free extracts of Nitrosomonas europaea

Summary The incorporation of [³⁵S]sulphate was followed into the washed cell suspensions of Nitrosomonas europaea. Thus bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine and methionine were found in the ethanol soluble fraction as well as in the residual hydrolysed protein fraction. Cysteic acid, methionine sulphoxide and methionine sulphone were detected in the residual protein. The reaction between sulphydryl groups and N-ethylmaleimide has been successfully used to stabilize the thiol compounds in cell-extracts and the derivatives thus obtained were separated by paper chromatography. As in other microorganisms, sulphate is first activated by ATP in Nitrosomonas before it is reduced. The formation of APS and PAPS has been studied. A pathway for the incorporation of [³⁵S]sulphate is proposed.Abbreviations POPOP 1,4-bis-(5-phenyloxazolyl-2)-benzene - PPO 2,5-diphenyloxazole - APS adenosine-5-phosphosulphate - PAPS adenosine-3-phosphate 5-phosphosulphate - ATP adenosine triphosphate - DNA-ase deoxyribonuclease - NEM N-ethylmaleimide - TCA trichloro-acetic acid - GSH glutathione     

The apparent turnover of mitochondria, ribosomes and sRNA of the brain in young adult and aged rats

—[¹⁴C] orotic acid and [³H]l -leucine were injected intraperitoneally into two groups of rats, aged 12 and 24 months, respectively. The apparent turnover of RNA and protein from several subcellular fractions was assessed by following the loss of label from these fractions with time. The curves for apparent turnover of all protein fractions from mitochondria were single exponential curves. Total mitochondrial protein from younger animals had a half-life of 26.8 days. Two protein subfractions, protein insoluble in cold perchloric acid and chloroform-methanol (residual protein) and protein soluble in chloroform-methanol (C–M protein) had similar half-lives: 26.3 and 26.1 days, respectively. For the older animals the half-lives were 23.5 days for total protein, 17.4 for residual protein and 30.4 for C–M protein. The difference between the two protein subfractions from mitochondria of the older animals suggests an age-associated deviation from the synchrony of synthesis and degration of proteins in this organelle. Further deviation from the unit concept of mitochondrial turnover was seen in the apparent turnover of mitochondrial RNA. Mitochondrial RNA had half-lives of 10.0 and 11.6 days for older and younger animals, respectively, with no significant difference between the groups. No age-associated difference was observed in the apparent turnover of sRNA. This fraction exhibited a double exponential turnover pattern; the first component in both cases had a half-life of about 5–8 days and the second component 13–16 days. Ribosomal RNA and protein from both older and younger animals exhibited multiexponential kinetics but both components, RNA and protein, within each age group appeared to turn over synchronously. Average values for apparent turnover of total ribosomes (RNA and protein) were 18.2 days for the older animals and 7.4 days for the younger animals. The age-associated difference was highly significant P < (0.001).     

In vitro Maintenance of the Trophonts and Sporozoites of Pyxinia crystalligera*

SYNOPSIS. In a study of in vitro maintenance of the trophonts and sporozoites of the cephaline gregarine, Pyxinia crystalligera, a basal medium consisting of salts, reducing agents, and antibiotics was supplemented by fetal bovine serum, serum protein fraction IV-4, or protein hydrolysates. Motility was used as the viability criterion. The tests were terminated when viability was ～30% of the original inoculum. Two-week survival of trophonts was observed with 0.75% protein fraction, 80% of organisms remaining viable at the end of 1 week. Similar results were noted with 2% casein hydrolysate. With lactalbumin hydrolysate and Casamino acids trophonts remained viable for 6 days, and with fetal bovine serum the results were similar to those obtained with lower concentrations of protein fraction IV-4. Sporozoites remained motile for up to 5 days in the basal medium supplemented by both protein fraction IV-4 and diluted haemocoelic contents of the host, Dermestes vulpinus, but only for 3 days or less in that supplemented by the protein fraction alone. The characteristic crystals of trophonts were dissolved or possibly utilized during in vitro maintenance. A new type of crystal, however, was observed in forms kept in the presence of higher protein hydrolysate concentrations. Smaller trophonts were more adaptable to maintenance than larger ones, for rapid depletion of paraglycogen and resorption of the deutomerite often was observed in the latter. Results of cytochemical tests for carbohydrates, neutral lipids, and proteins indicated that the trophonts were capable of paraglycogen synthesis in vitro.     

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.     

Studies of short-lived radicals in the gamma-irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography.

An aerated aqueous solution of uridine-5'-monophosphate was gamma-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radical which was the radiolytic product of the trapping reagent.