Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P₁₁₀, P₈₈, P_71,72, and P₂₃ had unique peptide maps. Evidence of a 27,000 dalton precursor to P₂₃ was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P₈₈ and P_71,72 increased from basal to new plateau levels in about 1.5 hours, while P₂₃ required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the metabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.     

Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues.

1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent 'chase' period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.     

Specific protein synthesis in cellular differentiation: VI. Temporal expression of chorion gene families in Bombyx mori strain C108

Detailed patterns of expression for putative members of 5 major chorion gene families have been obtained by separating labeled proteins using two-dimensional polyacrylamide gel electrophoresis. Proteins fall into 4 temporal cohorts called early, early middle, middle, and late on the basis of when they initiate and terminate synthesis. Proteins synthesized during the early, early middle, and late periods are highly synchronous, exhibiting abrupt onset times and relatively uniform termination times. Middle proteins begin synthesis in small groups at staggered times over a relatively long period, but most cease translation as the late proteins turn on. This data is correlated with a previous follicle staging system based on separation of newly synthesized chorion proteins by isoelectric focusing alone. The absolute timing of choriogenesis was determined in vivo, using trypan blue dye to mark vitellogenic follicles. The relative age difference between chorionating follicles was 2.2–2.6 hr; chorion biosynthesis took 4 days in all. These data are discussed in terms of patterns of activity of chorion gene families, the functions of the temporal cohorts, and regulation of the chorion multigene system.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Degradation of proteins in canavanine-grown yeast

Degradation of normal as well as canavanine proteins in growing yeast is suppressed by glucose. This suggests that the same mechanism may be operating in the catabolism in both cases. Degradation of normal and canavanine proteins is increased by disintegration of cell structure. Proteins synthesized in the presence of an amino acid analogue may not be degraded preferentiallyin vivo even when they are rather sensitive to endogenous proteolytic enzymes. Participant of the UNESCO postgraduate course onModern Problems in Biology.     

The nature of protein differentiation in the growing pea root

Abstract Sections of the growing root of pea (Pisum sativum) have been microdissected into stele, cortex and epidermis. Using labelled amino acids, two dimensional separations employing non-equilibrium pH gel electrophoresis (NEPHGE) and isoelectric focusing (IEF), and silver staining, the complexity of protein differences between the cortex and the stele has been assessed. Analyses commenced as cells in these two tissues appear in the meristem (0.7—1 mm from the tip) and continued up to 30 mm from the tip as they subsequently mature. From the earliest stages at which the cortex and stele can be distinguished and dissected apart the protein patterns differ substantially. However these tissue differences, involving over one third of the detected protein species, are almost all quantitative. Very few qualitative (i.e. tissue specific) proteins were detected. Many proteins also show quantitative stage-specific variation, detected using successively older root segments. In vitro translation studies involving isolated mRNA showed only a very limited stage-specific variation in translated proteins. This supports the notion that translational controls may contribute significantly to the development of these two tissue types.     

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”.     

Dibutyryl cAMP-induced protein changes in differentiating mouse neuroblastoma cells.

Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.     

Stimulation of proteinase biosynthesis by canavanine in streptococcus faecalis var. liquefaciens

l-Canavanine, an analogue of arginine, was found to stimulate the synthesis of an extracellular proteinase in Streptococcus faecalis var. liquefaciens. Cells grown in a synthetic medium containing 10(-4)m arginine and 10(-4)m canavanine produced almost twice as much proteinase as cells grown in 2 x 10(-3)m arginine alone; total growth was the same in both media. Hydrolyzed proteinase samples were analyzed for arginine and canavanine by means of paper chromatography and electrophoresis. Arginine, but not canavanine, was detected in the purified enzyme sample.     

Food control by applied biochemistry of marine organisms: Comparison of proteins and metabolites from fish and invertebrate muscle

Most fishery products consist of muscle tissue from fish and invertebrates. Differences in the molecular structure and in metabolism of muscles can be utilized to characterize and identify various seafood. Creatine and arginine were found to be useful for the differentiation between imitation crab/shrimp meat and real crustacean meat. Octopine served as an indicator for the meat of cephalopods and mussels. In order to identify the animal species of a fishery product, several electrophoretic methods were used. It depended on the type of product, whether sarcoplasmic or myofibrillar proteins were better suited. Raw products were best analysed by isoelectric focusing of sarcoplasmic proteins. Two types of sarcoplasmic calcium-binding proteins, parvalbumins of fish and soluble calcium-binding proteins of invertebrates, were especially useful for species identification. Due to their thermal stability, these proteins gave species-specific patterns for cooked products, too. Two other techniques were also investigated: urea gel isoelectric focusing, and sodium dodecyl sulphate — polyacrylamide gel electrophoresis. These methods were applied in the analysis of products where the sarcoplasmic proteins had been removed by washing steps, i.e. imitation crab meat made from surimi, and of other raw and cooked products. The myosin light chains gave protein patterns that were characteristic for many species. Paramyosin, which is absent from vertebrate muscle, indicated the presence of mollusc muscle. It was shown that the determination, of arginine kinase activity enabled differentiation between raw fish muscle and invertebrate muscles.     

Isolation of viable wheat male gametophytes of different stages of development and variations in their protein patterns

Procedures have been designed to isolate viable immature male gametophytes from wheat (Triticum aestivum L.) anthers of different stages of development. Maceration of anthers by a micro-blender allowed for the release of numerous intact vacuolate microspores. Tris-buffered media prevented tricellular pollen grains from bursting during the isolation procedure. Proteins from the undamaged male gametophytes have been analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A set of new proteins was detected at the onset of the second pollen grain mitosis. The results demonstrate the opportunity for studies on haploid gene expression at the translational level.     

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with ¹⁴C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2–1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.Participant in the UNESCO Postgraduate Course On Modern Problems in Biology and Microbial Technology.     

Exposure to arginine analog canavanine induces aberrant mitochondrial translation products,mitoribosome stalling,and instability of the mitochondrial proteome

Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Comparison of soluble proteins in juice and wine from Koshu grapes

Proteins were separated from Koshu grape juice and wine by precipitation with ammonium sulfate. The protein fractions were further fractionated by gel electrophoresis and gel isoelectric focusing, followed by amino acid analyses. The juice contained more than eleven protein fractions with molecular weights between 13,000 and 65,000, and their isoelectric points were between 3.6 and 10.5. The wine also contained more than eleven protein fractions with molecular weights between 21,000 and 65,000, while their isoelectric points were between 3.6 and 11.0. All the juice proteins and some major wine proteins were glycoproteins. The same three protein fractions were present in both juice and wine. The other juice proteins were lost during wine-making and thus, were not detected in the wine. About half of the proteins detected in the wine were not observed in the juice. Some juice proteins were bound to the flavonoid phenolics extracted from the wine and were removed as insoluble precipitates. There was specific interaction between wine flavonoids and juice proteins.     

Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Effects of canavanine on the secretion of plasma proteins by Hep G2 cells

Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins.     

Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing

We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.