Comparison by electrophoresis of proteins characteristic of the lateral line and skin of Xenopus laevis

In experiments aimed at determining acousticolateralis marker proteins, fractions of lateral-line organs and skin of Xenopus laevis were analyzed by one- and two-dimensional polyacrylamide-gel electrophoresis. A protein fraction of approximately 44K mol. wt (K = 1000 daltons) and isoelectric pH 6.3, consisting of at least two components, was enhanced in lateral-line neuromast tissue (containing hair cells) and was decreased in tactile organs and skin (lacking hair cells). This "neuromast-marker-protein" fraction had a mol. wt close to that of actin but was shown to be different from actin. Two other major proteins, at mol. wts 16 and 28K, were present in gels of skin and absent in gels of lateral-line tissue. These proteins were shown to be due to secretion of the amphibian granular glands and were designated "negative marker proteins".     

Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells.

Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system. It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site. Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene. beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons. Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides. Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.     

Structure, isolation, and protein composition of the pellicle of Sarcocystis muris cystozoites (Protozoa, Coccidia)

The molecular organization of the Sarcocystis muris cystozoite pellicle has been investigated by freeze-fracture electron microscopy and by electrophoresis of the proteins of isolated pellicles. Freeze-fracture revealed a highly ordered organization of the inner membrane complex similar to the one described in other coccidian zoites. Purification of pellicles was achieved by French Press homogenization followed by sucrose gradient floatation. Electron microscopy of the pellicle fraction demonstrated the partial preservation of the triple-membrane structure whereas freeze-fracture showed the disorganization of the particle arrangements of the inner membrane complex. The SDS-PAGE of the fraction revealed a complex protein composition with one major protein of 31,000 daltons, not labeled by lactoperoxidase-catalyzed surface iodination of living cystozoites.     

A Remeasurement of the Molecular Weights of T-Even Bacteriophage Substructural Proteins

T-even bacteriophage substructural proteins were studied by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that tail fibers are composed of two major proteins of 155,000 and 120,000 daltons molecular weight and four minor proteins of 51,000, 38,000, 27,000, and 23,000 daltons. Tail tubes were composed of one predominant protein of 18,500 daltons and one minor protein of 35,000 daltons molecular weight. Tubular polyheads obtained from a T4D amber mutant and by treatment of T4B-infected cells with L-canavanine were also examined, and no significant differences were noted in the molecular weight of the P23 protein.     

Purification and characterization of multiplication-stimulating activity (MSA) carrier protein

The rat liver cell line, BRL-3A, is known to produce a family of polypeptides referred to as multiplication-stimulating-activity (MSA). Serum-free conditioned medium from this cell line is a rich source for the purification of these somatomedin-like molecules. Somatomedins in serum, as well as MSA produced by BRL-3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000–70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.     

Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.     

Secreted amylolytic enzymes from Schwanniomyces occidentalis: purification by fast protein liquid chromatography (FPLC) and preliminary characterization

Amylolytic enzyme preparations are used extensively for the liquefaction and saccharification of starch in the production of ethanol and SCP (single cell protein). We report the first purification of two amylolytic enzymes from the yeast Schwanniomyces occidentalis using fast protein liquid chromatography (FPLC) in a two step process: size exclusion (Superose 12) followed by anion exchange (Mono Q). The procedure is amenable to direct scale up processes. The enzymes glucoamylase (E.C. 3.2.1.2) and alpha-amylase (E.C. 3.2.1.1) were found in the cell free supernatant of S. occidentalis when grown on a variety of carbon sources. The enzymes are substrate induced and catabolite repressed. Both amylolytic enzymes were purified from three separate culture broths containing either starch, maltose or cellobiose and their physical properties compared. Native molecular masses of glucoamylase and alpha-amylase were determined to be 122,000 +/- 28,000 daltons and 47,000 +/- 11,000 daltons, respectively, while subunit size was approximated at 143,000 +/- 2,000 daltons and 54,500 +/- 1,000 daltons, respectively. Both proteins are N-glycosylated with carbohydrate representing 10-15% of the total mass. The correlation of native mass and denatured subunit structure, while not identical due to slight aberrant behavior on gels and columns as a result of glycosylation, suggest that both proteins exist as monomeric polypeptides. Isoelectric points for both proteins under native conditions could not be determined since alpha-amylase failed to enter native polyacrylamide gels. However, a pI for glucoamylase of 6.2 +/- 0.2 (native) and a pI for alpha-amylase of 6.3 +/- 0.3 (in 6M urea) were determined. Glucoamylase and alpha-amylase specific activities (for the homogeneous proteins) were determined to be 48-67 x 10(3) units/mg and 214-457 x 10(3) units/mg respectively. We could find no apparent differences in either glucoamylase or alpha-amylase proteins obtained from three separate cultures which had been grown on different carbon sources. The purification method we have utilized is easily scaled up to larger protein concentrations, and provides a rapid procedure for analyzing and purifying these amylolytic enzymes.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.     

Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins

A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.     

Effects of cold, starvation and immobilization on the composition of acid-soluble nuclear proteins in chicken liver

Nuclear proteins soluble in 0.2 M sulphuric acid were isolated from the liver of three groups of hens subjected for 60 hours to starvation, immobilization or cold exposure. The obtained proteins were separated by means of one-dimensional and two-dimensional electrophoresis on polyacrylamide gel. It was observed that this exposure of the birds to stress caused no qualitative changes in liver nuclear proteins. Histones, histone-like proteins - M1, M2, M3, uM1, HMG 1 and 2 proteins, and a large group of non-histone protein fractions gave nearly identical patterns. However, several components of nuclear proteins were found whose quantity changed in the liver of the birds subjected to stress. These changes were observed in a protein with molecular weight about 27 000 daltons and two proteins weighing over 100 000 daltons.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Nucleotide sequence and organization of the transforming region and large terminal redundancies (LTR) of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) is a replication-defective acute leukemia virus, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. The genome of the AMV has undergone a sequence substitution in which a portion of the region normally coding for the "env" protein has been replaced by chicken cellular sequences. These latter sequences are essential for the transforming activity of the virus. We have determined the complete nucleotide sequence of this region. Examination of the AMV oncogenic sequence revealed an open reading frame starting with the initiation codon ATG within the acquired cellular sequences and terminating with the triplet TAG at a point 33 nucleotides into helper viral sequences to the right of helper-viral-cellular junction. The stretch of 795 nucleotides would code for a protein of 265 amino acids with a molecular weight of 30,000 daltons. The eleven amino acids at the carboxy terminus of such a protein would be derived from the env gene of helper virus.     

Cell-free translation of human pheochromocytoma messenger RNA yields protein(s) containing methionine-enkephalin

Cell-free translation of messenger RNA extracted from a human pheochromocytoma yields protein(s) of apparent molecular weight >70,000 daltons which contain the pentapeptide methionine-enkephalin. It is estimated that 0.8–1.0% of the total pheochromocytoma mRNA codes for the methionine-enkephalin-containing protein, based on percent incorporation of [³⁵S]methionine into methionine-enkephalin during cell-free translation. These results demonstrate that human pheochromocytoma mRNA contains the message for a high-molecular weight methionine-enkephalin-containing protein or proteins, presumably the methionine-enkephalin precursor molecule(s).     

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.     

Metal-protein binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) during short term cadmium stress

Cd-binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) have been analysed in response to the administration of a single dose of Cd ions. Four main components, with apparent molecular weights of 3-4, 12, 20 and greater than 70 K daltons, have been isolated from soluble cell fractions. The variation in molar metal/protein ratios of these components is discussed in terms of the uptake, transfer and detoxification of Cd in the hepatopancreas. A model is proposed which describes Cd flux at the subcellular level and indicates that metallothionein-like proteins may mediate in this process.     

The nucleotide sequence of the transforming HindIII-G fragment of adenovirus type 5 DNA. The region between map positions 4.5 (HpaI site) and 8.0 (HindIII site).

The nucleotide sequence of the region between map positions 4.5 (HpaI-site) and 8.0 (HindIII-site) of adenovirus type 5 (Ad5) DNA has been determined. This stretch of DNA is part of the transforming HindIII-G fragment, which is 2809 nucleotides long. The sequenced segment was found to have a long open reading frame for protein biosynthesis, starting 23 nucleotides from the HpaI site and extending all the way to the HindIII-G site, which could code for a protein of at least 44 000 daltons. The possible correlation beteen the coding capacity of the HindIII-G fragment and the "transforming" proteins specified by it will be discussed in the light of the recent data on the splicing of early mRNAs.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Three structural polypeptides coded for by minite virus of mice, a parvovirus.

Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step.     

Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis

The cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis were isolated from parasporal crystals and subsequently separated from each other. The proteins were separated by gel filtration chromatography and their molecular weights were estimated by both gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. The apparent molecular weights of the mosquitocidal protein and the cytolytic protein were estimated to be 65,000 daltons and 28,000 daltons, respectively.     

The seed proteins of chick pea: Comparative studies ofCicer arietinum,C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatum Ladiz. andC. echinospermum Davis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.