Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

Mouse mammary tumor virus (MuMTV) was purified from two cell lines (GR and Mm5MT/c1), and the genomic RNA was isolated and translated in vitro in cell-free systems derived from mouse L cells and rabbit reticulocytes. The major translation product in both systems was a protein with the molecular weight 77,000. Several other products were also detected, among them a 110,000-dalton and in minor amounts a 160,000-dalton protein. All three polypeptides were specifically immunoprecipitated by antiserum raised against the major core protein of MuMTV (p27), but they were not precipitated by antiserum against the virion glycoprotein gp52. Analysis of the in vitro products by tryptic peptide mapping established their relationship to the virion non-glycosylated structural proteins. The 77,000-dalton polypeptide was found to be similar, if not identical, to an analogous precursor isolated from MuMTV-producing cells. Peptide mapping of the 110,000-dalton protein shows that it contains all of the methionine-labeled peptides found in the 77,000-dalton protein plus some additional peptides. We conclude that the products synthesized in vitro from the genomic MuMTV RNA are related to the non-glycosylated virion structural proteins. Polyadenylic acid-containing RNA from MuMTV-producing cells also directed the synthesis of the 77,000-dalton polypeptide in the L-cell system. If this RNA preparation was first fractionated by sucrose gradient centrifugation the 77,000-dalton protein appeared to be synthesized from mRNA with a sedimentation coefficient between 25 and 35S.     

Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 μM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH₃- and NH₂OH-dependent O₂ consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii.

In Klebsiella pneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer.     

Coping with iron limitation: a metabolomic study of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Iron (Fe) is a key element for all living systems, especially for photosynthetic organisms because of its important role in the photosynthetic electron transport chain. Fe limitation in cyanobacteria leads to several physiological and morphological changes. However, the overall metabolic responses to Fe limitation are still poorly understood. In this study, we integrated elemental, stoichiometric, macromolecular, and metabolomic data to shed light on the responses of Synechocystis sp. PCC 6803, a non-N₂-fixing freshwater cyanobacterium, to Fe limitation. Compared to Synechocystis growing at nutrient replete conditions, Fe-limited cultures had lower growth rates and amounts of chlorophyll a, RNA, RNA:DNA, C, N, and P, and higher ratios of protein:RNA, C:N, C:P, and N:P, in accordance with the growth rate hypothesis which predicts faster growing organisms will have decreased biomass RNA contents and C:P and N:P ratios. Fe-limited Synechocystis had lower amounts Fe, Mn, and Mo, and higher amount of Cu. Several changes in amino acids of cultures growing under Fe limitation suggest nitrogen limitation. In addition, we found substantial increases in stress-related metabolites in Fe-limited cyanobacteria such antioxidants. This study represents an advance in understanding the stoichiometric, macromolecular, and metabolic strategies that cyanobacteria use to cope with Fe limitation. This information, moreover, may further understanding of changes in cyanobacterial functions under scenarios of Fe limitation in aquatic ecosystems.     

Siderophore-mediated uptake of iron in Azotobacter vinelandii

Azotobacter vinelandii produces two siderophores, N,N'-bis-(2,3-dihydroxybenzoyl)-L-lysine (azotochelin) and a yellow-green fluorescent peptide (azotobactin), under iron-limited growth conditions. 55Fe uptake was not observed until the substantial nonspecific binding of 55Fe to the cell surface was eliminated by the addition of 10 mM sodium citrate to the uptake medium. Citrate alone did not promote rapid 55Fe uptake in A. vinelandii, nor did it induce Fe-repressible outer membrane proteins. Siderophore-mediated 55Fe uptake appeared biphasic, with both the initial rapid and ensuing slower uptake being energy dependent. The purified siderophores demonstrated the same uptake pattern as the Fe-limited culture supernatant fluid, but either individually or in combination accounted for less than the total 55Fe uptake activity found in the latter. The purified siderophores appeared to be sensitive to acid, but the inhibition of 55Fe uptake was in fact caused by salt generated during neutralization. Similar 60% inhibition of 55Fe uptake activity was caused by the addition of 40 mM Na+, K+, Li+, or Mg2+ salts to the uptake medium. Ammonium was less inhibitory than the latter ions. 55Fe uptake mediated by azotobactin was more sensitive to added NaCl than was that mediated by azotochelin. Neither the chelation of iron nor the stability of the ferrisiderophore was affected by added NaCl.     

The presence of ADP-ribosylated Fe protein of nitrogenase in Rhodobacter capsulatus is correlated with cellular nitrogen status

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.     

Influence of quorum sensing and iron on twitching motility and biofilm formation in Pseudomonas aeruginosa

Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl₃ concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl₃ that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.     

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.     

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)- ATPase function.     

Synthesis and deposition of spore coat proteins during sporulation of Bacillus megaterium

Rabbit (anti-spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fraction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000-dalton protein, which is a major protein, did not cross-react with any other major (36,000, 22,000, 19,500, and 17,500-dalton) proteins. Specific antibody to the 22,000-dalton protein did not cross-react with the 48,000, 36,000, 19,500, 17,500, and 16,000-dalton proteins, but did cross-react with the 44,000, 25,000, and 12,000-dalton proteins.     

Recovery of competence in calcium-limited Azotobacter vinelandii.

Azotobacter vinelandii cells required 0.5 mM calcium in the iron-limited competence induction medium. This requirement also was fulfilled by strontium, but not by magnesium. Cells pregrown in competence medium lacking calcium rapidly recovered competence with the addition of 0.5 mM calcium, provided they were suspended in the growth supernatant. A 60,000-dalton glycoprotein (pI 5.10) present in competent or incompetent culture supernatants participated in calcium-mediated competence recovery. Cells grown in calcium-limited medium appeared to have leaky cell envelopes and released a diverse array of proteins into the culture supernatant and into distilled water washes of the cells, seven of which appeared to be more dominant in competent cells. Two distilled water washes of cells grown in calcium-limited medium did not prevent calcium-mediated recovery of competence in the culture supernatant. Four to six distilled water washes removed a competence-specific protein (pI 5.19) and prevented calcium-mediated recovery of competence in the culture supernatant.     

Head Proteins from T-Even Bacteriophages II. Physical and Chemical Characterization

Three classes of structural head proteins, having molecular weights of 43,000, 18,000, and 11,000 daltons, were isolated from the T-even bacteriophages and were characterized. Based on electrophoretic studies, the 43,000-dalton class contained one major protein and one (or two) minor components, the 18,000-dalton class contained two protein components, and the 11,000-dalton class contained one major component. The N-terminal residues for the 43,000- and the 11,000-dalton classes were alanine, and the N-terminal residues for the 18,000-dalton class were methionine and alanine. Of the three classes of proteins, the 18,000-dalton proteins were the most acidic, whereas the 11,000-dalton proteins were the most basic. The amino acid composition of the 11,000-dalton class revealed that methionine and cysteine were absent and lysine, histidine, and tryptophan content was higher in the 11,000-dalton class than in the other two classes of proteins. Estimates of the relative number of the three classes of structural proteins were made and indicated that there were between 1,600 and 2,000 subunits of the 43,000-dalton proteins, 100 to 200 of the 18,000-dalton proteins, and 1,000 to 1,500 of the 11,000-dalton proteins. Evidence was presented that the 43,000-dalton proteins and the 11,000-dalton proteins readily formed aggregates with themselves but not with each other. The significance of these interactions to the structure of the T-even phage head was discussed.     

Regulation of molybdate transport by Clostridium pasteurianum.

The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH3, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of bells in absence of transport synthesis. Carbamyl phosphate (greater than or equal to 15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 mug/ml) is added just before the exhaustion oNH 3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if WO42- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO42- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period.     

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.     

Identification and characterization of cellular targets for tyrosine protein kinases

Protein kinases associated with the transforming proteins of a number of retroviruses are specific for tyrosine. Several proteins in cells transformed by these viruses are phosphorylated at tyrosine. We have now identified three unrelated abundant nonphosphorylated cellular proteins of 46,000, 39,000 and 28,000 daltons in chick embryo cells, which are the unphosphorylated forms of phosphotyrosine-containing proteins and thus are substrates for tyrosine protein kinases. By two-dimensional gel analysis, we have found that the 46,000-dalton protein exists in two unphosphorylated forms of which the more acidic is a minor species. This latter form is phosphorylated, chiefly at serine, in both normal and transformed cells, generating a yet more acidic species. In transformed but not normal cells, the major form is phosphorylated at tyrosine and serine, yielding a fourth isoelectric variant. The 46,000-dalton unphosphorylated protein has been partially purified, and antiserum to it recognizes all four isoelectric variants of the protein. The 39,000-dalton protein has two unphosphorylated forms of which the more acidic is a minor species. The major form is phosphorylated at tyrosine and serine in transformed cells only. The 39,000-dalton unphosphorylated protein has been partially purified, and antiserum raised to it recognizes all three isoelectric variants. The 28,000-dalton protein has a single phosphorylated form which contains serine in normal cells, but both serine and tyrosine in transformed cells.