Divergence of the two albumins of X. laevis. Evidence for the glycosylation of the major 74K albumin

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis, differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated that 74K albumin gene sequences exist but that they are not present in the amplified DNA library. A X.laevis genomic library was therefore constructed and screened for albumin genes without amplification. Both 68K and 74K albumin gene sequences were isolated. Recombinants containing 74K albumin gene sequences grew extremely poorly and this probably explains why the 74K albumin sequences were ot isolated from the amplified library. Characterisation of the cloned DNA indicates that there is one sequence coding for the 68K albumin but two different sequences coding for the 75K albumin.     

Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

Dexamethasone and estrogen regulate Xenopus laevis albumin mRNA levels

The regulation of albumin mRNA levels by steroid hormones was examined in a primary Xenopus liver culture system. In the absence of exogenous steroid hormone, albumin mRNA levels declined rapidly in culture. Dexamethasone is required for preservation of albumin mRNA levels in culture and effects a greater than 10 fold induction of albumin mRNA in cultures maintained in steroid hormone-free medium. Estrogen can override the effect of dexamethasone and elicits a decline of greater than 80% in albumin mRNA levels. Our cDNA clones of the mRNAs encoding the two 74,000 dalton and one 72,000 dalton cellular albumins allowed us to show that all three albumin mRNAs were down regulated by estrogen.     

Identification and characterization of the avian erythroblastosis virus erbB gene product as a membrane glycoprotein

Avian erythroblastosis virus causes erythroid leukemia and sarcomas in chickens. The viral oncogene responsible for these diseases, erb, is divided into two regions known as erbA and erbB, and recent evidence suggests that it is the erbB gene that is responsible for the transforming activity. From rats bearing avian erythroblastosis virus-induced sarcomas, we have obtained antisera which are specific for the erb gene products. Using such antisera, we have been able to characterize the erbB gene product as a 68,000 molecular weight protein. Pulse-chase and cell-free in vitro translation experiments show that the initial product is a 62,500 dalton protein which is initially modified to a 66,000 dalton protein, and then further modified to a 68,000 dalton form. These modifications could be shown to be associated with glycosylation and phosphorylation. Cell fractionation experiments revealed that the 66,000 and 68,000 dalton proteins were located in cell membrane fractions, and immunofluorescence results showed the erbB gene product to be expressed on the cell surface.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Alterations in the synthesis of proteins in hepatocytes of rainbow trout fed cyclopropenoid fatty acids

Hepatocytes from rainbow trout reared on a diet containing cyclopropenoid fatty acids were analyzed for alterations in protein composition and synthesis by double label experiments. Both cytosolic and microsomal hepatocyte fractions were investigated. In the cytosolic fraction, the synthesis of proteins in the range of 68,000 to 74,000 daltons were significantly decreased. The identity of these proteins remains uncertain. A pronounced depression in both the mass and apparent synthesis of a 200,000 to 240,000 dalton microsomal protein was also observed. Immunoblotting with antibodies raised against goose acetyl-CoA carboxylase and avidin-peroxidase staining suggest that this protein is acetyl-CoA carboxylase. Moreover, synthesis of this protein as well as mass of the protein in cyclopropenoid fatty acid-fed fish were less than 20% of that found in control fish.     

Diphtheria toxin-receptor interaction: A polyphosphate-insensitive diphtheria toxin-binding domain

Inositol hexaphosphate, and other polyphosphates, inhibit diphtheria toxin-mediated cytotoxicity by binding to the toxin at a highly cationic site called the P site and preventing toxin binding to cell surface receptors. The binding of diphtheria toxin to a solubilized cell surface glycoprotein (150,000 daltons) is also inhibited by these polyphosphates. Treatment of this 150,000 dalton diphtheria toxin-binding cell surface glycoprotein with papain yielded an 88,000 dalton and a 74,000 dalton diphtheria toxin-binding glycoprotein whose binding to toxin was no longer inhibited by inositol hexaphosphate. This result suggests a model of diphtheria toxin-receptor interaction in which the toxin receptor possesses one binding site which interacts with the P site of the toxin in a $\text{polyphosphate-sensitive}$ fashion, and another binding site (located within the papain-derived 74,000–88,000 dalton glycoproteins) which can interact with the toxin at a site distinct from the P site (the X site) in a $\text{polyphosphate-insensitive}$ fashion. This X site-receptor interaction may be involved in the binding of CRM proteins that bind to the toxin receptor but that do not bind polyphosphates, or it may be involved in the entry process of the toxin.     

The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis.

Serum from hamsters bearing group C adenovirus-induced tumors can be divided into two classes: first, a broad spectrum serum that contains antibodies to several early adenovirus proteins, immunoprecipitated from virus-infected cell extracts, with molecular weights of 72,000, 58,000, 44,000 and 17,000 daltons; and second, a narrow spectrum serum that contains antibodies to the 58,000 dalton protein from virus-infected cell extracts. Both types of sera have been used to immunoprecipitate specifically the 58,000 dalton protein from a type 2 adenovirus-transformed hamster cell line and a type 2 adenovirus-SV40 nondefective hybrid (Ad2⁺ND-1) transformed hamster cell line. In addition, the broad spectrum serum immunoprecipitates or co-precipitates a late adenovirus protein of 120,000 daltons from virus-infected, but not virus-transformed cells.Peptide maps of the 120,000 dalton antigen and the virus hexon structural protein (120,000 daltons) demonstrate that these proteins are closely related. The 72,000 dalton antigen has been shown to be the adenovirus single-strand-specific DNA binding protein. Peptide maps of this 72,000 dalton antigen demonstrate that it contains all the peptides found in the 44,000 dalton antigen. The 72,000 dalton antigen contains two additional peptide fragments not detected in the 44,000 dalton protein, indicating that this 44,000 dalton antigen is a proteolytic breakdown product of the 72,000 dalton protein. The 58,000 dalton adenovirus tumor antigen has a peptide map which is completely distinct from the 120,000, 72,000 and 44,000 dalton proteins. These data demonstrate that the 58,000 dalton antigen is chemically distinct from the 72,000–44,000 dalton early adenovirus proteins.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

In vitro translation of mushroom tyrosinase

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.     

High molecular weight forms of relaxin in pregnant sow ovaries

The existence of a prohormone for relaxin has been investigated by purification from extracts of pregnant sow ovaries. Radioimmunoassay of column fractions from large scale extraction of frozen pregnant sow ovaries detected two species of high molecular weight relaxin immunoactivity besides the 6,300 dalton relaxin. On further purification, these two species were resolved into three forms with apparent molecular weights of 19,000, 13,000 and 10,000 daltons respectively. Each was biologically active and could be converted by trypsin to the 6,300 dalton relaxin. They may represent intermediates of relaxin.     

Immunochemical identity of the high and low molecular weight forms of Galapagos marine iguana hemoglobin.

1. Two forms of Galapagos marine iguana methemoglobin, with molecular weights of 140,000 and 70,000 daltons, were identified in iguana RBC lysates by Sephadex G-200 molecular sieve fractionation. 2. The 140,000 dalton ferric hemoglobin was isolated by DEAE-Sephadex A-50 ion-exchange chromatography and found to be pure by electrophoretic and immunological criteria. 3. Immunochemical analyses revealed the high and low molecular weight hemoglobins to be antigenically identical.     

Proteins crosslinked to messenger RNA by irradiating polyribosomes with ultraviolet light.

A set of proteins crosslinked to L-cell mRNA by irradiating polyribosomes with 254 nm ultraviolet light has been identified. 35S-methionine-labeled crosslinked mRNA-protein complexes were isolated by chromatography on oligo(dT)-cellulose under conditions that prevented non-covalent binding of proteins to RNA and to the column. After enzymatic removal of the RNA the proteins were analyzed in sodium dodecylsulfate/polyacrylamide gels. Six proteins having molecular weights of 98,000, 78,000, 75,000, 68,000, 62,000, and 52,000 were crosslinked to mRNA whether intact polyribosomes or EDTA- released mRNA-protein complexes were irradiated. Digestions with specific RNAases and chromatography on oligo(dT)-cellulose were used to show that a protein of 78,000 daltons was the only one crosslinked to poly(A), and the other proteins were crosslinked to sequences other than poly(A). However, a 78,000 dalton protein was also crosslinked to a sequence other than poly(A).     

Serum amyloid A (SAA) protein-interaction with itself and serum albumin.

Serum amyloid A (SAA) protein is a 12,000 dalton protein that exists in serum under physiologic conditions as an 85,000 dalton complex and under certain conditions, as a 170,000 dalton component. To study the reason for this finding, the behavior of 125I-SAA was studied in the presence of cold SAA and several serum proteins. SAA caused a shift of some of the radioactivity to the region of albumin. Addition of normal human serum or albumin caused a shift of a significant fraction of the radioactivity to a peak eluting slightly ahead of albumin (80.000 daltons). This interaction could be blocked by the addition of cold SAA. No shift was noted when IgG or Bence Jones proteins were added. Thus, it appears that low molecular SAA protein has a tendency to aggregate with itself and to bind to albumin but not to human IgG or Bence Jones proteins.     

Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation.

Following transformation of chick embryo fibroblasts (CEF) by avian RNA tumor viruses, two membrane polypeptides with apparent molecular weights of 90,000 and 75,000 daltons have been found to be increased (Stone, Smith and Joklik, 1974). We find that this alteration in membrane proteins is not directly related to transformation.The 90,000 and 75,000 dalton proteins are present in increased amounts in a 3T3 fibroblast mutant (AD6) defective in glycoprotein synthesis. Feeding the mutant N-acetylglucosamine, a metabolite that bypasses the metabolic block, restores the amount of these two proteins to the levels found in normal cells. The 75,000 dalton protein is markedly reduced, and the 90,000 dalton protein disappears and is replaced by a fully glycosylated derivative with a molecular weight of 92,000 daltons.Two glucose derivatives, glucosamine and 2-deoxyglucose, are known to interfere with the glycosylation process. The addition of these substances to normal CEF and 3T3 cells specifically induces the accumulation of the 90,000 and 75,000 dalton membrane polypeptides.Finally, the deprivation of glucose for 24–48 hr also induces the synthesis of the 90,000 and 75,000 dalton polypeptides in normal fibroblasts. The induction of these two proteins by glucose starvation suggests that they have a role in glucose utilization.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Human term placenta contains transforming growth factors

Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.