Fluorography of tritium-labelled proteins in immunoelectrophoresis

A method is described for detecting 3H-labelled proteins in immunoelectrophoretic systems performed on agarose gels. The method is based on the incorporation of a polyacrylamide gel into the agarose gel after the electrophoresis. This mixed gel has the characteristics of a polyacrylamide gel, making it possible to use fluorography as has been described for polyacrylamide gels. The applicability of the fluorography method is demonstrated by analyzing 3H-labelled human serum albumin and 3H-labelled pig intestinal brush border proteins by quantitative immunoelectrophoresis.     

Concomitant determination of absolute values of cellular protein amounts,synthesis rates,and turnover rates by quantitative proteome profiling

Two-dimensional gel electrophoresis of protein fractions isolated from (35)S-radiolabeled cells provides qualitative information on intracellular amounts, (35)S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and (35)S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for (35)S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.     

Changes in the incorporation of labelled amino acids into proteins and homogenates of the brains of rats that have suffered acute hypoxia in the antenatal period]

The authors studied C14-leucine and S35-methionine incorporation into the brain tissue homogenates and protein from different parts of the brain of rats subjected to intrauterine hypoxia. Depression of protein synthesis in certain brain structures, particularly in the hyppocampus was observed alongside with the stimulation of the amino acid incorporation into proteins of the other parts of the brain. Changes of the amino acid penetration into tissue homogenates fialed to correlate with the rate of their incorporation into proteins in separate structures of the brain. Experimental results pointed to disfunction in the protein metabolism intensity and in the blood-brain barrier system occurring during the late ontogenesis in rats surviving the intrauterine hypoxia.     

The effect of psychotropic substances (aminazin, majeptil, trisedil) on protein synthesis in different regions of the rat brain]

Experiments were conducted on rats; a study was made of methionineS35 incorporation into the sum total proteins isolated from various portions of the brain after a single administration of chlorpromazine, majeptil and tricedil. A generalized depression of protein synthesis in all the structures, except the medulla oblongata, followed chlorpromazine administration in one and three hours. A stimulating effect is characteristic of majeptil in the majority of the brain portions. The action of tricedil was accompanied by reduction of methionine-S35 incorporation into the proteins of the majority of the brain structures and by an increase in its incorporation into the olfactory lobes. As supposed, changes in the protein synthesis in individual structures of the brain served as an important link in the action mechanism of psychotropic preparations on the organism.     

The effect of amino acids on protein metabolism as measured in long-term experiments in immature brain explants

In a study of a system suitable for investigating long-term effects on brain protein metabolism, we measured amino-acid incorpration into isolated immature brain explants incubated under sterile conditions up to ten days. Measurements of changes in total proteins, total DNA, cell number during the experiments, and 14C-thymidine incorporation measurements indicated no significant net growth; new cell formation was below 5% in a 5-day period; therefore, amino-acid incorporation was mainly due to protein turnover. The rate of incorporation in our immature brain preparation was similar to that of the adult brain in vivo: by ten days about one-half of the tissue protein turned over. The label incorporated was released in subsequent incubations with cold amino acids. Such release occurred in all subcellular fractions examined. Incorporation was fairly stable; at temperatures below 30 degrees C it rapidly declined, but it was not affected when phenylalanine or the branched chain amino acids (leucine, isoleucine, valine) were elevated in the incubation medium. Brief exposure to low amino-acid media had no effect; longer exposure resulted in tissue damage. Our model system indicates that overall brain protein turnover is not sensitive to such variations in the level of most amino acids, which may occur under various conditions. Protein metabolism of the nervous system occurs at a high rate. A recent long-term labeling method (Lajtha, Latzkovits, and Toth, 1976) gave a best fit to incorporation curves by assuming two compartments for adult brain proteins, one of which (about 6%) has a half-life of 15 hr and the other (94%) has a half-life of ten days. The disappearance of protein-bound label with time under conditions in which all proteins were previously labeled indicated that most, possibly all, proteins in brain are in a dynamic state (Lajtha and Toth, 1966). Incorporation of amino acids was found in all proteins and structures that have been studied to date; myelin proteins previously thought less active are also metabolized at a significant rate (Sabri, Bone, and Davison, 1974; Lajtha, Toth, Fujimoto, and Agrawal, 1977). We have fairly extensive information available in addition to turnover studies about the mechanisms of protein synthesis in brain (Roberts, 1971); protein breakdown was also studied in some detail (Marks and Lajtha, 1971). In contrast to our knowledge about protein metabolism under physiological equilibrium conditions, our information about alterations during functional demands or pathological conditions is scanty. Although a significant amount of work has been reported, largely because of technical difficulties the results are difficult to interpret unequivocally. The present report represents our effort to address some of the obstacles: to develop a system in which influences on long-term incorporation can be studied...     

Biochemical and Autoradiographical Determination of Protein Synthesis in Experimental Brain Tumors of Rats

The rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5-3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma-derived) and endogenous (proteolysis-derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60-70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 +/- 16.0 nmol/g of tissue/min in brain tumor, and 17.2 +/- 4.2 and 9.7 +/- 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.     

Nicotine-induced changes in the metabolism of specific brain proteins

The effect of acute and chronic nicotine on the metabolism of specific brain proteins was examined by measuring incorporation of labeled valine into protein, with densitometric scanning of proteins resolved by gel electrophoresis. Acute and chronic administration of nicotine (0.4 mg/kg per 30 min for 2 hours, s.c., or 0.5 mg/kg per 30 min for 5 days (Alzet mini-pump implanted subcutaneously) reduced incorporation of [¹⁴C]valine administered by approximately 6–7%. The results with chronic nicotine administration indicated a lack of tolerance for this effect of nicotine. Mecamylamine, a nicotinic ganglionic antagonist, does not seem to block the inhibition of protein synthesis. Small increases in protein content were observed in a high- and a low-molecular-weight region of SDS-polyacrylamide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective bands. Results are consonant with selective effects of nicotine on the synthesis or degradation of specific brain proteins.Special Issue Dedicated to Dr. Abel Lajtha.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

The effect of morphine tolerance and dependence on cell free protein synthesis

A cell free system consisting of polyribosomes and pH 5 factors of the cytosol was isolated from mouse brain. This system actively promoted the incorporation of radiolabeled amino acids into protein in vitro. Addition of exogenous morphine to a cell free protein synthetic system isolated from chronically morphinized, placebo treated, or naive mouse brain had no effect on the relative synthetic capacity of the system. In addition, morphine did not alter the response to a synthetic mRNA, polyuridylic acid. However, both the polyribosomes and pH 5 factors isolated from chronically morphinized mouse brain were more effective in promoting amino acid incorporation into protein relative to the corresponding fractions from placebo treated mice. Acrylamide gel electrophoresis of the proteins in the incubation mixture showed the increased amino acid incorporation was the result of a general quantitative increase in the specific activity of all of the proteins synthesized by the cell free system.     

Photoaffinity labelling of the serotinin carrier protein in platelets and brain synaptosomes

Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focussing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [³H]azidoimipramine.     

Phosphorylation in vivo of four basic proteins of rat brain myelin

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H₃³²PO₄. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [³²P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.     

Measurement of in vivo incorporation of L-methionine-35S into cerebral proteins by quantitative autoradiography in freely-moving or anesthetized rats

Incorporation of L-35S-methionine in brain proteins was measured by quantitative autoradiography in the rat. This model was checked by biochemical procedures. In awake freely moving rat, methionine incorporation rates vary from 36 to 258 nmol/100g/min. During anaesthesia, protein synthesis in brain decreases (30 to 50%).     

Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [³⁵S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.     

De novo synthesis of peroxidase in cotton ovule culture medium

The biosynthesis of cotton ( Gossypium hirsutum L. 'Stoneville 208') peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [³⁵S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [³⁵S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.     

Alpha-synuclein gene deletion decreases brain palmitate uptake and alters the palmitate metabolism in the absence of alpha-synuclein palmitate binding

Alpha-synuclein is an abundant protein in the central nervous system that is associated with a number of neurodegenerative disorders, including Parkinson's disease. Its physiological function is poorly understood, although recently it was proposed to function as a fatty acid binding protein. To better define a role for alpha-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or alpha-synuclein gene-ablated mice with [1-(14)C]palmitic acid (16:0) and assessed fatty acid uptake and turnover kinetics in brain phospholipids. Alpha-synuclein deficiency decreased brain 16:0 uptake 35% and reduced its targeting to the organic fraction. The incorporation coefficient for 16:0 entering the brain acyl-CoA pool was significantly decreased 36% in alpha-synuclein gene-ablated mice. Because incorporation coefficients alone are not predictive of fatty acid turnover in individual phospholipid classes, we calculated kinetic values for 16:0 entering brain phospholipid pools. Alpha-synuclein deficiency decreased the incorporation rate and fractional turnover of 16:0 in a number of phospholipid classes, but also increased the incorporation rate and fractional turnover of 16:0 in the choline glycerophospholipids. No differences in incorporation rate or turnover were observed in liver phospholipids, confirming that these changes in lipid metabolism were brain specific. Using titration microcalorimetry, we observed no binding of 16:0 or oleic acid to alpha-synuclein in vitro. Thus, alpha-synuclein has effects on 16:0 uptake and metabolism similar to those of an FABP, but unlike FABP, it does not directly bind 16:0; hence, the mechanism underlying these effects is different from that of a classical FABP.     

Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Synthesis of the myelin proteolipid protein in the developing mouse brain

Mice ranging in age from 16 to 44 days were injected intracerebrally with ³H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of ³H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.