Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase

The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.     

A Simple Procedure for the Isolation of Brain Actin

Actin has been isolated from bovine brain by forming an actin-containing gel from acetone powders. Solubilization of the gel and chromatography on Sephadex G150 produces actin in good yield. Acetone powders of crude pellets of brain homogenates obtained in the isolation of tubulin produces about 80% of the actin isolated from acetone powder of whole brain. The procedure therefore can be used to isolate both proteins from the same brain.     

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.     

Acetone precipitation--an improved procedure for the isolation of soybean agglutinin

Isolation of soybean agglutinin (SBA) by the salt fractionation involves excessive amounts of (NH4)2SO4. We have found that SBA could be fractionally precipitated from an aqueous extract by adding acetone (40% final concentration). It is stable under these conditions for minimum 2 h at 5 degrees C and 25 degrees C. Incorporating these results, an improved procedure for the isolation of SBA has been developed. The SBA isolated by this method is obtained in better yield, has 6000 HU/mg protein and is identical to that isolated by the (NH4)2SO4 method as ascertained by chromatographic and electrophoretic comparisons and hapten inhibition assays.     

An Algorithm that Predicts the Viability and the Yield of Human Hepatocytes Isolated from Remnant Liver Pieces Obtained from Liver Resections

Isolated human primary hepatocytes are an essential in vitro model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.     

Isolation and certain properties of human pituitary prolactin]

A simple method of isolation of highly purified prolactin from acetonated preparations of anterior hypophysial lobes is described. The new method permits to obtain higher (about 10-fold) yields of the hormone, as compared to those obtained using previously described methods. Prolactin was extracted by acid aqueous acetone and was subsequently purified of extract by fractionation with acetone and NaCl and by isoelectric precipitation. The final stage of the hormone purification involved gel-filtration through Sephadex G-200; prolactin yield was 400 microgram per 1 hypophysis. The lactogenic activity of the hormone is 14 MU/mg; the sequence of N-terminal amino acid residues of prolactin is as follows: NH2-Leu-Pro-Ile-x-Pro-Leu(?)-Gly-Ala-.     

Characterization of an Inhibitor of Hepatic Cholesterogenesis Present in Hepatic Microsomes

An inhibitor of hepatic cholesterol synthesis present in hepatic microsomes can be solubilized either by an acetone or an ethanol powder preparation. Other methods such as methanol and chloroform:methanol powder preparations and treatment with EDTA do not solubilize the factor.

The factor appears to be proteinaceous since its activity is lost after exposure to proteolytic enzymes and heat treatment. In addition, the inhibitor does not require a phospholipid for activity.

This inhibitor is stable for long periods (60 hrs.) at room temperature and can be isolated in good yield from liver maintained at 4°C for 8 hours postmortem.     

Preparative isolation of adult human liver metallothionein isoforms

Preparative human liver metallothionein (MT) isolation is described. MT was saturated with cadmium to follow MT purification spectrophotometrically instead of by metal content and to increase the stability of the protein. A concentrated, MT-rich fraction of the liver cytosol was prepared by selective organic solvent (acetone or acetone/methanol) fractionation. Conventional gel filtration and ion-exchange chromatographies resolved two MT isoforms, MT-I and MT-II. When needed, purification of MT from other low-molecular weight proteins was further increased by gel filtration chromatography at zero ionic strength, i.e., in distilled water. Reversed-phase high performance liquid chromatography of both MT isoforms resolved further peaks sharing MT properties not only from the MT-I but also from the MT-II ion-exchange isoform. The results show that it is feasible to perform a human liver MT isolation from an entire human liver with a reasonable laboratory capability.     

StreptoTag: a novel method for the isolation of RNA-binding proteins

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.     

Evaluation of protein extraction protocols for 2DE in marine ecotoxicoproteomics

In ecotoxicoproteomics, an accurate and reproducible extraction of proteins is a critical step for 2DE analysis and further protein identification using MS. The criteria for the assessment of protein extraction quality include protein yield, protein spots resolved in a 2DE gel, matched protein spots in replicate gels, reproducibility, and compatibility with MS. In this work, we evaluated three protein extraction systems, straightforward lysis buffer, trichloroacetic acid–acetone, and TRIzol reagent with some modifications, for the protein extraction from three animal species including mussel Mytilus galloprovincialis, flounder Paralichthys olivaceus, and polychaete Nereis diversicolor used in marine ecotoxicology. Our results indicated that these methods could extract significantly different protein profiles. The method using TRIzol reagent resulted in the most matched protein spots resolved in four replicate 2DE gels and highest reproducibilities for the gill of M. galloprovincialis and liver of P. olivaceus. However, a modified trichloroacetic acid–acetone solvent system was best for the whole soft tissue of N. diversicolor. This work provides the fundamental information of the extraction quality of protein extraction protocols from different marine animals, which may facilitate the selection of a suitable protein extraction protocol for ecotoxicoproteomics.     

The recovery of nitrocellulose-bound protein

Conditions for the electroelution of protein from polyacrylamide gels to nitrocellulose and its subsequent recovery have been examined. A procedure is described whereby soluble material suitable for further analysis can be obtained. Nitrocellulose-bound protein is dissolved in acetone. It can be precipitated from solution in the presence of a carrier such as Polybrene in a form that can subsequently be solubilized in good yield.     

High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins

Abstract

An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.     

Rapid one-step purification of ferritin from cell-free translation systems

A method is described for the one-step affinity purification of ferritin from other proteins synthesized in an in vitro wheat germ protein synthesizing system programmed with rat liver mRNA. The peptide products released from the polysomes were chromatographed on an antiferritin-Sepharose 4B affinity column. The ferritin isolated in this manner was judged pure by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The recovery of the purified protein from the antibody column ranged from 83 to 88%. Antibody affinity columns can be arranged sequentially to allow the isolation of numerous proteins from one protein synthesis reaction mixture. This method of isolation is applicable to any protein for which antibody is available.     

Acetone Precipitation—An Improved Procedure for the Isolation of Soybean Agglutinin

Isolation of soybean agglutinin (SBA) by the salt fractionation involves excessive amounts of (NH₄) ₂ SO ₄. We have found that SBA could be fractionally precipitated from an aqueous extract by adding acetone (40% final concentration). It is stable under these conditions for minimum 2 h at 5°C and 25°C. Incorporating these results, an improved procedure for the isolation of SBA has been developed. The SBA isolated by this method is obtained in better yield, has 6000 HU/mg protein and is identical to that isolated by the (NH₄)₂ SO ₄ method as ascertained by chromatographic and electro-phoretic comparisons and hapten inhibition assays.     

Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons

We have failed to detect the presence of mannose-6-phosphate in the oligosaccharide moiety of glycoproteins from pea (Pisum sativum L. cv Burpeeana) cotyledons using an assay system sensitive to 10 picomoles of mannose-6-phosphate. We were also unable to demonstrate any retention of glycosidase activity from pea seedlings and pea cotyledons on Sepharose-coupled phosphomannosyl receptor proteins isolated from bovine liver which were, however, able to retain phosphomannosylated hexosaminidase purified from Dictyostelium discoideum secretions. Furthermore, although Sepharose-coupled phosphomannosylated hexosaminidase from Dictyostelium was able to bind phosphomannosyl receptors from bovine liver we were unable to detect the retention of any protein from acetone powder extracts of pea seedlings or from endoplasmic reticulum-associated proteins of pea cotyledons.

Based on this collective evidence we conclude that mannose-6-phosphate does not appear to play a role in the targeting of hydrolytic enzymes from the endoplasmic reticulum to the protein bodies in pea cotyledons.

     

Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer.

The S-layer of Caulobacter is a two-dimensional paracrystalline array on the cell surface composed of a single protein, RsaA. We have established conditions for preparation of stable, soluble protein and then efficient in vitro recrystallization of the purified protein. Efficient recrystallization and long range order could not be obtained with pure protein only, though it was apparent that calcium was required for crystallization. Recrystallization was obtained when lipid vesicles were provided, but only when the vesicles contained the specific species of Caulobacter smooth lipopolysaccharide (SLPS) that previous studies implicated as a requirement for attaching the S-layer to the cell surface. The specific type of phospholipids did not appear critical; phospholipids rather different from those present in Caulobacter membranes or archaebacterial tetraether lipids worked equally well. The source of LPS was critical; rough and smooth variants of Salmonella typhimurium LPS as well as the rough form of Caulobacter LPS were ineffective. The requirement for calcium ions for recrystallization was further evaluated; strontium ions could substitute for calcium, and to a lesser extent, cobalt, barium, manganese and magnesium ions also stimulated crystallization. On the other hand, nickel and cadmium provided only weak crystallization stimulation, and zinc, copper, iron, aluminum ions, and the monovalent potassium, sodium, and lithium ions were ineffective. The recrystallization could also be reproduced with Langmuir-Blodgett lipid monolayers at an air-water interface. As with the vesicle experiments, this was only successful when SLPS was incorporated into the lipid mix. The best method for RsaA preparation, leading to apparently monomeric protein that was stable for many months, was an extraction with a low pH aqueous solution. We also achieved recrystallization, albeit at lower efficiency, using RsaA protein solubilized by 8 M urea, a method which allows retrieval of protein from inclusions, when expressed as heterologous protein in Escherichia coli or when retrieved as shed, precipitated protein from certain mutant caulobacters. In summary, the clarification of recrystallization methods has confirmed the requirement of SLPS as a surface attachment component and suggests that its presence in a membrane-like structure greatly stimulates the extent and quality of S-layer formation. The in vitro approach allowed the demonstration that specific ions are capable of participating in crystallization and now provides an assay for the crystallization potential of modified S-layer proteins, whether they were produced in or can be secreted by caulobacters.     

Isolation of Protein uH2A Using a one Step Preparative Gel Electrophoresis

Abstract

A one step electrophoretic procedure for the isolation of protein uH2A has been devised which may improve the overall yield. The improvement involves elimination of intermediate steps which might result in the decrease of the yield. The method may serve as an alternate to the conventional methods and can also be used successfully for the isolation of several different proteins.     

Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.