Lack of interaction between hormonal proteins and synthetic carrier ampholytes (Ampholine)

Using ¹⁴C-labeled Ampholine, radioiodinated and unlabeled human growth hormone, and ovine prolactin, no significant degree of interaction could be detected by polyacrylamide gel electrophoresis and gel chromatography. Protein binding to excess Ampholine was negligible. Ampholine binding to excess protein was below the detection limit of 0.2 moles of Ampholine per mole of protein. Commercial Ampholine contains large molecular weight components that cannot be separated by gel chromatography from proteins in the 20–30,000-molecular weight range.     

Cytokinin-modulated gene expression in excised pumpkin cotyledons

Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)⁺ mRNA isolated from cotyledons incubated with 10⁻⁴ molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10⁻⁹ to 10⁻⁴ molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

High-affinity folate binding in human liver membranes

High-affinity binding of³H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel^® AcA 44 chromatography of solubilized membranes saturated with³H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

The effect of SDS on protein zone dispersion in polyacrylamide gel electrophoresis

The zone dispersions of the reduced subunit of β-lactoglobulin B and its derivative with sodium dodecyl sulfate (SDS) were measured during polyacrylamide gel electrophoresis (PAGE) using the apparatus for continuous optical scanning at 280 nm. The ratio of apparent diffusion coefficients (D′) of the reduced subunit of β-lactoglobulin B (1.71 × 10^?6 cm²/s) and of its SDS-derivative (7.1 × 10^?7 cm²/s) was found to be 2.4 under the conditions of PAGE (pH 10.4, 0.015 ionic strength, 1°C, 4 mA/cm² current density, 50 μg protein load, 10% T gel) used. This is nearly twice the value of 1.3 predicted, under the assumption of sphericity for these protein molecules, on the basis of the binding of 1.4 g of SDS per gram of protein. It is postulated that the increment in zone sharpness (decrease in apparent diffusion coefficient) over that predicted by SDS binding alone is a general property of SDS-proteins providing gel electrophoresis in SDS-containing buffers with a resolving power larger than that obtained in the absence of the detergent.     

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)⁺ RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient electrophoresis-SDS polyacrylamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis, both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1–2 h). Among them, three were repressed markedly     

Binding of Escherichia coli heat-stable enterotoxin and rise of cyclic GMP in COLO 205 human colonic carcinoma cells

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [¹²⁵I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a K_d of 0.5×10⁻¹⁰ M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [¹²⁵I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Cadmium speciation studies in the intestine of Lumbricus terrestris by electrophoresis of metal proteins complexes

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO₃)₂ supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (¹⁰⁹Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.     

Carbohydrate composition of rat hepatocyte nuclear membrane as compared to normal, Morris hepatoma 7800, and phenobarbital-induced microsomal membranes

The composition of the nonhistone nuclear proteins of rat liver, brain, thymus, and kidney has been analyzed by isoelectric focusing in polyacrylamide gel. Approximately 20–30 components were separated with a wide range of isoelectric points (pl) in the 3- to 10-pH region.Different extraction procedures applied to liver nuclei removed protein mixtures with similar components present in varying amounts. 8 m Urea 50 mm phosphate, pH 7.6, was the most successful and removed most of the nonhistone protein.The thiol groups of proteins extracted from the nuclei of liver, brain, thymus, and kidney with 8 M urea, 50 mM phosphate were labeled with [¹⁴C]N-ethylmaleimide. Although there was a slight variation in the overall thiol content of these tissue proteins, separation of the mixture by isoelectric focusing and SDS polyacrylamide gel electrophoresis showed complex patterns indicating greater heterogeneity than was apparent from the Coomassie blue dye binding.     

Identical 110,000 dalton phosphoproteins in nuclei and polyribosomes of a rapidly growing rat hepatoma

Nuclei and free polyribosomes from rat hepatoma tissue were incubated $\text{in vitro}$ in the presence of [³²P] ATP. 0.2 N HCl-soluble nuclear proteins and polyribosomal proteins binding to oligo (dT) cellulose were prepared. A 110 kd phosphoprotein was separated by SDS polyacrylamide gel electrophoresis from each of the samples. The phosphoprotein was analysed using limited digestion with protease V8 in a second SDS gel. The patterns obtained for stained peptides and phosphopeptides were completely identical for both preparations. Thus significant amounts of the 110 kd protein appear to be present, as genuine constituents, in both nuclei and polyribosomes.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis,amino acid analysis,and surface labeling

Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using ¹²⁵I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.     

Purification of leukocytosis-promoting substance from bovine parotid gland extract

A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 · 10⁴, and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.     

Identification of neighboring protein pairs in the Escherichia coli 30 S ribosomal subunit by crosslinking with methyl-4-mercaptobutyrimidate

The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.     

In vitro binding of labeled 20-methylcholanthrene to a specific cytosol-protein

In vitro binding kinetics and specificity of [20-³H]methylcholanthrene ([³H]MC) interaction with mouse epidermal cytosol in the presence or absence of microsomal metabolizing systems were investigated. After an incubation period of 30 roin at 22°, the samples were dialyzed, subjected to gel filtration, ultrafiltration, and analyzed by 7 % basic polyacrylamide gel electrophoresis. A major portion of the binding appeared in a single peak on the gel which had the same mobility as bovine or mouse serum albumin. In vitro competition by other hydrocarbons or by promoters for binding of MC to this cytosol receptor protein showed an impressive correlation to carcinogenic and promoting activities. Essentially 100% of the MC bound to cytosol without a microsomal metabolizing system was non-covalent. However, binding Of MC to cytosol in the presence of microsomes plus reduced NADPH was party covalent which has previously been reported by Grover and Sims and Meunier and Chaveau. When bovine (BSA) or mouse serum albumin (MSA) was incubated with radioactive MC in the presence of competing non-radioactive carcinogens or promoters, little or no inhibition of binding was found. The finding that both a powerful tumor promoter and a strong carcinogen are competitors for the specific MC binding to the cytosol receptor protein indicates that it may represent a critical interaction for the promoting stage of chemical carcinogenesis in mouse skin.     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 μM cadmium chloride (Cd^r) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [³⁵S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cd^r lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [³⁵S]Cysteine-labelled protein eluted at a on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7–17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound ¹⁰⁹Cd²⁺.