Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40.

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.     

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons. The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%. Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins. Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass. Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.     

Comparison of Two Human Papovaviruses with Simian Virus 40 by Structural Protein and Antigenic Analysis

The proteins of simian virus 40 (SV40) and two human papovaviruses, the hemagglutinating BK virus and the non-hemagglutinating DAR virus, were analyzed and compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The virions of SV40 and DAR contain seven proteins. By molecular weight analysis the constituent proteins of SV40 and DAR are identical. Approximately 84% of the viral protein has a molceular weight of 45,000. The major protein of BK virus is 3,000 to 5,000 daltons lighter than the major proteins of SV40 and DAR viruses. The five most rapidly migrating proteins of BK virus are indistinguishable by molecular weight analysis from the corresponding proteins of SV40 and DAR viruses. Radial immunodiffusion and immunoelectrophoresis of whole virus gave lines of identity between SV40 and DAR when reacted with SV40 antibody. SV40 antiserum tested against BK virus and BK antiserum tested against SV40 virus showed no reactivity by complement fixation, immunodiffusion, or immunoelectrophoresis.     

Purification and properties of cellulase from Phaseolus vulgaris

Cotyledons of germinating kidney beans contain two forms of a carboxy methyl cellulase which can be separated by ammonium sulfate fractionation and isoelectric focusing. The two cellulases are similar in their molecular weight but differ in isoelectric points, pH and temperature optimum, pH and temperature stability and sensitivity to thiol inhibitors and metal ions. One cellulase (isoelectric point 4.8) has been purified 100-fold to give a major protein band on acrylamide gel electrophoresis.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

露花叶片PEP羧化酶的纯化及某些分子特性

经硫酸铵分部沉淀,DEAE-纤维素(DE52),DEAE-Sephadex A-50,SephacrylS-200和二次羟基磷灰石等柱层析,从露花叶片中分离得到纯化63.9倍、电泳均一的磷酸烯醇式丙酮酸羧化酶。此酶的天然分子量经聚丙烯酰胺梯度凝胶电泳测定为260kD,经SephadexG-200凝胶过滤法测定为240kD。用SDS-聚丙烯酰胺梯度凝胶电泳测得酶的亚基分子量为115kD,表明此酶是个二同聚体。此酶的等电点为PI=5.6。免疫双扩散的结果表明此酶与高梁PEPG的抗原决定簇呈部分同一性。     

Physicochemical characterization of a crystallin from the squid lens and its comparison with vertebrate lens crystallins

A crystallin was isolated from the homogenate of the Squid (Loligo pealii) lens by gel filtration on a Sepharose CL-6B (2.5 X 170 cm) column. Biochemical characterization showed it is a dimeric protein with a molecular weight of (5.1 +/- 0.4) X 10(4) and a Stokes' radius of 26A. Electrophoresis on a cellulose acetate membrane indicated it is a basic protein with an isoelectric point higher than 8.6. High resolution two-dimensional gel in 8 M urea/2% NP-40 resolved this crystallin into 6 charge isomers, each with a major subunit of molecular weight 29,000 daltons and a minor subunit of 27,000 daltons in a molar ratio of 3:1. The extreme susceptibility of the protein to denaturation and precipitation even at low temperature hampered further characterization of this crystallin under nondenaturing conditions. Amino acid analysis indicated it contains an unusually high content of methionine (12.8 mol%) which may have some bearing on the instability of this crystallin in vitro. Biochemical comparison of the squid crystallin with mammalian lens crystallins shows that it is a crystallin distinguishable from all reported vertebrate lens crystallins. A detailed study of this protein may shed light on the evolution of lens crystallins in general.     

Isolation and Characterization of Two Basic Internal Proteins from the T-Even Bacteriophages

Two species of basic internal proteins were found in osmotic shock supernatant solutions of bacteriophages T4B, T4D, T2H, T2L, and T6. The major species of protein isolated had a molecular weight of approximately 21,000 daltons, whereas the minor protein molecular weight was near 9,500 daltons. The two protein species exhibited unique isoelectric points and amino acid compositions. The 21,000-dalton protein of T2L showed major electrophoretic and compositional differences from the other 21,000-dalton proteins isolated. Similarities between the 21,000-dalton proteins and phage lysozyme are discussed.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.     

Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.     

Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells

In this report we describe the biochemical characterization of neutrophil and monocyte p8 and p14. Together the two proteins comprise approximately 45% of cytosolic protein in neutrophils and approximately 40-fold less in monocytes. They fractionated together in several chromatographic procedures and were found to exist as a noncovalently associated complex with a stoichiometry of 1:1, named p8,14. Cross-linking experiments showed p8,14 to form heterodimers under conditions simulating the cytosol. An apparent molecular mass of 35,000 daltons was obtained for the p8,14 complex in molecular sizing experiments which suggests the presence of modifications or distinctive structural features. Two major forms of p14 can be identified by two-dimensional gel electrophoresis, both of which form heterodimers with p8. The lower molecular weight variant of p14 lacks Cys-3 (Met-Thr-Cys-Lys-Met...) suggesting that differing translational start sites account for these two forms of p14. A protocol has been devised for the rapid purification of milligram quantities of p8 and p14 from neutrophil cytosol using fast-protein liquid chromatography.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.     

Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40.

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.     

The structural basis of ankyrin function. I. Identification of two structural domains

The structure of ankyrin, a major linking protein between spectrin and the erythrocyte membrane, was analyzed after restricted proteolytic digestion at 0 degree C. By the use of two-dimensional peptide mapping, we found that tryptic digestion of ankyrin (1 h, 0 degree C) resulted in the production of two nonoverlapping peptides of molecular weights 82,000 and 55,000. The 82,000-dalton peptide had a basic isoelectric point (7.9) and was remarkably sensitive to further proteolytic digestion; after 24 h at 0 degree C, trypsin completely digested this peptide into fragments too small to detect by gel electrophoresis. The 55,000-dalton peptide was neutral (isoelectric point = 6.9-7.2) and more resistant to further proteolytic cleavage. After a 24-h digestion with trypsin at 0 degrees C, the 55,000-dalton peptide was cleaved into two complementary fragments of molecular weight 32,000 and 15,000. Analysis of phosphorylated ankyrin indicated that the phosphates were exclusively found in these two complementary peptides. By comparison with larger fragments, we were able to align the constituent peptides of ankyrin and propose a low resolution model. Ankyrin appears to be a bipolar molecule containing a basic domain of 82,000 daltons and a neutral phosphorylated domain of 55,000 daltons.     

Structural Proteins of Adenovirus-Associated Virus Type 3

Three major structural proteins were found in adenovirus-associated virus (AAV) type 3H virions which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the polypeptides were determined to be approximately 66,000 (VP1), 80,000 (VP2), and 92,000 (VP3). The component having a molecular weight of 66,000 comprised about 80% of the total virion protein in the major AAV-3H particle, and the other two components comprised about 10% each. Proteins of the same molecular weight were found in the minor dense AAV-3H virion, but the 80,000- and 92,000-molecular-weight components were present at about one-half the concentration. The AAV-3H virion contains about 72 molecules of VP1 and 8 and 7 molecules of VP2 and VP3, respectively.     

Correlation between genetic loci and structural differences in the capsid proteins of polyoma virus plaque morphology mutants.

Two plaque morphology variants of polyoma virus (A-2 and 208) showed marked differences in agarose gel electrophoresis of the whole particles, isoelectric focusing of the major capsid protein VP1 (45,000 daltons) and three tryptic peptides (A, B and C) of VP1. No major difference in apparent molecular weight on NaDodSO₄ gels, amino acid composition or carbohydrate detectable by Schiff staining was revealed between the capsid proteins of the two viruses.Correlations have been made between phenotype, portions of the primary amino acid sequence of VP1 and the physical map of polyoma virus DNA by analysis of this protein from large plaque A-2 virus, minute plaque 208 virus and large plaque 208 virus selected after marker rescue with a fragment of polyoma virus DNA generated by the Hpa II restriction enzyme. The interrelationship of these properties was established by taking advantage of the observations of Miller, Cooke and Fried (1976)that heterozygous markers present on heteroduplex DNA are found in 100% of selected progeny and in only 50% of unselected progeny.All five marker rescued isolates selected for large plaque morphology showed only two A-2-specific characters, the absence of peptide C in tryptic maps of VP1 and the aggregation of VP1 on isoelectric focusing. The other four characters which distinguish A-2 and 208 were present or absent in 40–60% of the five isolates, which is close to the expected 50% for unselected markers. Three of the four A-2-specific characters (the presence of peptide A, absence of peptide B and isoelectric point of VP1) have been found to occur coordinately in the marker rescued isolates. The fourth character (electrophoretic mobility of virus particles in agarose gels) segregated independently.The techniques used in this study should find wide application in correlating primary amino acid sequence, nucleotide sequence and phenotype in other systems.