Concentrations Of Polypeptides Research Articles

Peptide recognition by a synthetic receptor at subnanomolar concentrations.

This paper describes the discovery and characterization of a dipeptide sequence, Lys-Phe, that binds to the synthetic receptor cucurbit[8]uril (Q8) in neutral aqueous solution with subnanomolar affinity when located at the N-terminus. The thermodynamic and structural basis for the binding of Q8 to a series of four pentapeptides was characterized by isothermal titration calorimetry, NMR spectroscopy, and X-ray crystallography. Submicromolar binding affinity was observed for the peptides Phe-Lys-Gly-Gly-Tyr (FKGGY, 0.3 μM) and Tyr-Leu-Gly-Gly-Gly (YLGGG, 0.2 μM), whereas the corresponding sequence isomers Lys-Phe-Gly-Gly-Tyr (KFGGY, 0.3 nM) and Leu-Tyr-Gly-Gly-Gly (LYGGG, 1.2 nM) bound to Q8 with 1000-fold and 170-fold increases in affinity, respectively. To our knowledge, these are the highest affinities reported between a synthetic receptor and an unmodified peptide. The high-resolution crystal structures of the Q8·Tyr-Leu-Gly-Gly-Gly and Q8·Leu-Tyr-Gly-Gly-Gly complexes have enabled a detailed analysis of the structural determinants for molecular recognition. The high affinity, sequence-selectivity, minimal size of the target binding site, reversibility in the presence of a competitive guest, compatibility with aqueous media, and low toxicity of Q8 should aid in the development of applications involving low concentrations of target polypeptides.

Study on mutagenesis of high protease producing thermophilic bacteria by UV-DES and hydrolysis of excess sludge

Excess sludge a resource-rich organic waste, can be solubilized by thermophilic enzymes to extract proteins and anaerobic digestion of methane production to reduce sludge and use resources. At the same time, it can improve new ideas to relieve the pressure of the energy industry. To solve the problems of low enzyme-producing activity of wild strains and low hydrolysis rate of ES, this study utilized UV-DES compound mutagenesis to screen dominant mutant strains under optimized conditions, which were added to ES hydrolysis reaction system. The results showed that UV-DES mutagenesis could effectively improve the ability of S-TE and promote the hydrolysis of ES. The protease activity of the mutant strain KT16 obtained by screening was 46.7% higher than that of the original strain DC8. KT16 effectively degraded insoluble organic matter, reduced the average particle size of ES, and increased the concentrations of extracellular proteins, polypeptides, and amino acids to 1731.8, 521.3, and 510 mg·L-1, respectively. This study will provide a reference for the resource utilized.

Preparation of reductive polypeptides from fresh placentas of dairy cows.

In order to prepare reductive polypeptides from the placenta of dairy cows’ fresh placentas from healthy Chinese Holstein cows were obtained and homogenized. Response surface model was established to optimize the hydrolysis condition for the extraction of the placental polypeptides. Specifically, the placental tissue homogenate was treated with both trypsin and pepsin for 348 min and 329 min; at 35.00% and 35.75% of substrate concentration; with an enzyme-substrate ratio of 3.33% and 3.92%, respectively, based on the models. The treated samples were then demineralized and freeze-dried to obtain the hydrolyzed polypeptides. In order to identify the molecular mass distribution and reducibility of polypeptides, matrix-assisted laser desorption ionization (MALDI) and Prussian blue methods were used. The concentrations of placental polypeptides after hydrolysis by trypsin or pepsin were 5.52% and 5.97%, respectively; the vitamin C (Vit C) equivalents were 36.26 μg mg-1 or 61.15 μg mg-1, respectively. Both groups showed intensity peaks of MALDI patterns in the range of 300 - 400 Da, and polypeptides hydrolyzed by pepsin had higher Vit C equivalent anti-oxidant activity than trypsin hydrolyzed polypeptide, suggesting that the proteins in the placental tissues were hydrolyzed to di-peptides and tri-peptides completely. In conclusion, both trypsin and pepsin hydrolysis performed well in preparation of reductive polypeptides from the fresh placentas of dairy cows; while, pepsin is more effective than trypsin. The primary reductive ingredients may be the oligopeptides with molecular mass less than 1000 Da.

Effects of ultra-high pressure treatment on angiotensin-converting enzyme (ACE) inhibitory activity, antioxidant activity, and physicochemical properties of milk fermented with Lactobacillus delbrueckii QS306

The present study investigated the influence of ultra-high pressure (UHP) treatment on angiotensin-converting enzyme inhibitory (ACEI) activity and quality of milk fermented with Lactobacillus delbrueckii QS306 after storage. By varying treatment pressure, duration of pressure treatment, and duration of fermentation, optimal process parameters for the UHP treatment of milk fermented with QS306 to enhance ACEI activity were found to be 400 MPa, 10 min, and 48 h, respectively. The degree of ACE inhibition of the fermented milk was 91.63 ± 0.02%. After UHP treatment, ACEI activity, apparent viscosity, concentrations of polypeptides and volatile aromatic substances, umami, and richness had increased significantly, bitterness and astringency were significantly reduced, and antioxidant properties were maintained. In addition, UHP fermented milk maintained a high level of ACEI activity and good quality during storage. Thus, the data represent a valuable reference for improving the storage quality of fermented milk and research for the future development of dairy products with high ACEI activity.

Limitations of the fasting proinsulin to insulin ratio as a measure of β-cell health in people with and without impaired glucose tolerance.

The fasting proinsulin to insulin ratio is elevated in people with type 2 diabetes and has been suggested as a marker of β-cell health. However, its utility in discriminating between individuals with varying degrees of β-cell dysfunction is unclear. Proinsulin has a very different half-life to insulin and unlike insulin does not undergo hepatic extraction prior to reaching the systemic circulation. Given these limitations, we sought to examine the relationship between fasting and postprandial concentrations of β-cell polypeptides (proinsulin, insulin and C-peptide) in people with normal and impaired glucose tolerance in differing metabolic environments. Subjects were studied on two occasions in random order while undergoing an oral challenge. During one study day, free fatty acids were elevated (to induce insulin resistance) by infusion of Intralipid with heparin. Proinsulin to insulin and proinsulin to C-peptide ratios were calculated for the 0-, 30-, 60- and 240-minute time points. Insulin action (Si) and β-cell responsivity (Φ) indices were calculated using the oral minimal model. The fasting proinsulin to c-peptide or fasting proinsulin to insulin ratios did not differ between groups and did not predict subsequent β-cell responsivity to glucose during the glycerol or Intralipid study days in either group. Among nondiabetic individuals, the fasting proinsulin to insulin ratio is not a useful marker of β-cell function.

The role of autocatalysis on the chemical diversity of the prebiotic ocean of early Earth

AbstractThe spontaneous formation of catalytic polypeptides of various lengths in a primordial ocean endowed with a source of amino acids from micrometeorites was investigated and found to be sufficient to induce the transformation of potential substrates under the assumption of a high propensity of the environment to catalyse the formation of the peptide bond. This work aims to include in this picture the effect of autocatalysis, i.e. the ability of a polypeptide with a specific length to promote the formation of the peptide bond. Once the formation of an autocatalytic species is attained, the concentrations of the polypeptides, substrates and products of reaction exhibit a time-dependent rate of formation and undergo a catastrophic change. While in the absence of autocatalysis the concentrations of polypeptides are stationary and the formation of reaction products is limited by the proper frequency λ, autocatalysis induces a steady growth of the concentrations of polypeptides and a 100 − 105-fold increase of reaction products at t = ω−1<0.46 Gyr, with a subsequent linear growth in time according to the law u/z0 = 1+s(ω−1+t)/z0, provided the autocatalytic species be active with length fewer than 70 amino acid units. A relationship was found between the catalytic ability of the environment (expressed by the ratio η/ηh of the rate coefficient for peptide bond formation to the corresponding rate coefficient for hydrolysis) and the time of the sharp increase of the concentration of both the polypeptides and their products of transformation. Although the formation of autocatalytic polypeptides is able to rapidly induce a sharp increase in the concentration of both polypeptides and their products of transformation, the crucial formation of the first autocatalytic polypeptides relies on the ability of the environment to promote the formation of the peptide bond. The value of the ratio η/ηh, constrained by the available time for chemical evolution to values bordering the catalytic activity of present-day enzymes, suggests that the correlation between the presence of water and the formation of a complex chemistry should be taken with caution.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

BackgroundFibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved.MethodsThe MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively.ResultsBoth of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity.ConclusionsPolypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Latex allergy: the sum quantity of four major allergens shows the allergenic potential of medical gloves

Assessment of allergenic potential of medical devices made of natural rubber latex (NRL) requires the measurement of concentrations of specific allergenic proteins or polypeptides eluting from rubber. Four NRL allergens (Hev b 1, 3, 5, and 6.02) were quantified in all medical glove brands marketed in Finland in 1999, 2001, and 2003 (n = 208) by a capture enzyme immunoassay. The results were compared with those obtained from previous nationwide market surveys, using a skin prick test-validated human IgE-based ELISA-inhibition method. A high overall correlation (r = 0.87, 95% CI 0.83-0.90) emerged between the sum values of the four allergens(microg/g glove) and IgE-ELISA inhibition (allergen units, AU/ml, 1 : 5 diluted glove extract). The sum of four allergens when set at 0.15 microg/g discriminated 'low allergenic' (<10 AU/ml) from 'moderate- to high-allergenic' (>/=10 AU/ml) gloves at a sensitivity of 0.93 (95% CI 0.85-0.98) and specificity of 0.90 (95% CI 0.83-0.94). When the sum was below the detection limit (0.03 microg/g) all gloves belonged to the previously defined low-allergen category. By comparing the sum concentration of four selected NRL allergens with results obtained in human IgE-ELISA inhibition, it was possible set a cut-off level (0.15 microg/g) below which virtually all gloves contain low or insignificant amounts of allergens, and can be considered as low allergenic. At different cut-off-points, one could calculate the likelihood of a given glove to belong to the previously defined low, moderate or high allergen categories.

Reversible Deficiency of Antimicrobial Polypeptides in Bacterial Vaginosis

Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human beta-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases.

Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

Physiological importance of endosomal acidification: potential role in proximal tubulopathies.

In recent years, there have been significant advances in our understanding of the molecular mechanisms relating proximal tubule abnormalities to the pathogenesis of renal Fanconi syndrome. This review focuses on the role of intra-endosomal acidification-machinery proteins (V-ATPase, CLC-5, NHE-3), as well as apical receptors (megalin and cubilin), in the receptor-mediated endocytosis pathway and in the pathogenesis of proximal tubulopathies. Animal models, including CLC-5 and megalin knockout mice, cubilin-deficient dogs and cadmium-toxicity studies in rats, have shed light on defects leading to low-molecular-weight proteinuria. In particular, the important contribution of defective endosomal acidification and membrane-protein recycling to the pathogenesis of the Fanconi syndrome has emerged from these studies. These observations, together with recent findings in patients with Dent's disease, Lowe's syndrome, autosomal-dominant idiopathic Fanconi syndrome and Imerslund-Grasbeck disease, show that the proteinuria of the Fanconi syndrome is more generalized than previously suspected. High concentrations of polypeptides, including hormones, vitamin-binding proteins and chemokines in urine from these patients and animals may play an important role in the progressive renal failure that is associated with the syndrome. The molecular mechanism of proximal tubule protein reabsorption, which is defective in renal Fanconi syndrome, includes a crucial role for endosomal acidification-machinery proteins, in particular the V-ATPase and CLC-5 chloride channels, in the trafficking and acidification-dependent recycling of apical membrane proteins, including the endocytotic receptors megalin and cubilin. An increased understanding of the roles of V-ATPase and CLC-5 in proximal tubule endosomal acidification, in the regulation of the megalin/cubilin-mediated endocytosis pathway and finally in the pathogenesis of human Fanconi syndrome will help in the devising of appropriate strategies for therapeutic intervention for this disorder.

An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays, which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.

Lengthening the Second Stalk of F1F0 ATP Synthase in Escherichia coli

In Escherichia coli F(1)F(0) ATP synthase, the two b subunits dimerize forming the peripheral second stalk linking the membrane F(0) sector to F(1). Previously, we have demonstrated that the enzyme could accommodate relatively large deletions in the b subunits while retaining function (Sorgen, P. L., Caviston, T. L., Perry, R. C., and Cain, B. D. (1998) J. Biol. Chem. 273, 27873-27878). The manipulations of b subunit length have been extended by construction of insertion mutations into the uncF(b) gene adding amino acids to the second stalk. Mutants with insertions of seven amino acids were essentially identical to wild type strains, and mutants with insertions of up to 14 amino acids retained biologically significant levels of activity. Membranes prepared from these strains had readily detectable levels of F(1)F(0)-ATPase activity and proton pumping activity. However, the larger insertions resulted in decreasing levels of activity, and immunoblot analysis indicated that these reductions in activity correlated with reduced levels of b subunit in the membranes. Addition of 18 amino acids was sufficient to result in the loss of F(1)F(0) ATP synthase function. Assuming the predicted alpha-helical structure for this area of the b subunit, the 14-amino acid insertion would result in the addition of enough material to lengthen the b subunit by as much as 20 A. The results of both insertion and deletion experiments support a model in which the second stalk is a flexible feature of the enzyme rather than a rigid rod-like structure.

Carboxylase genes of Sulfolobus metallicus.

Carbon dioxide limitation of Sulfolobus metallicus resulted in increased cellular concentrations of polypeptides that were predicted to be biotin carboxylase and biotin carboxyl-carrier-protein components of a protein complex. These polypeptides were coeluted from a native polyacrylamide gel and were estimated at 19 and 59 kDa after separation by denaturing gel electrophoresis. Their encoding genes were identified, sequenced and shown to code for polypeptides of 18,580 and 58,235 Da with similarities to biotin carboxyl carrier proteins and biotin carboxylases, respectively. The genes overlapped at the second of two stop codons that terminated the carboxylase gene. A third gene occurred on the opposite strand, 293 bp upstream of the biotin carboxylase gene. Its deduced amino acid sequence was similar to those of carboxyl transferase subunits of carboxylase enzymes, in particular to those of the propionyl-CoA carboxylases. It is proposed that the three described genes could encode the key enzyme complex responsible for carbon dioxide fixation during autotrophic growth of the thermoacidophilic archaea.

The Concentrations of Some Gut Polypeptides Are Elevated During Lactation in Ruminants

Circulating concentrations of both glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1(7-36)amide (GLP-1) were determined in dry and lactating sheep. Both polypeptides were significantly higher in lactating animals compared with dry. The half-life of both in plasma was determined by intravenous injection of GIP or GLP-1 in both dry and lactating animals, but neither peptide showed a significantly different value during lactation. Thus, the increased circulating concentrations during lactation must result from increased secretion, probably resulting from the increased feed intake during lactation. Serum insulin concentrations were significantly lower during lactation, which poses the question of how lactating animals maintain lower circulating insulin levels in the presence of higher levels of insulinotropic agents.

Correlations of Plasma Concentrations of β-Cell Polypeptides with pathologic features of Insulinomas

To assess the correlations between plasma concentrations of b-cell polypeptides (insulin, C peptide) and size, multiplicity, and malignancy of insulinomas. We retrospectively reviewed the medical records of 55 consecutively treated patients at our institution with surgically confirmed insulinoma. The 29 female and 26 male patients ranged from 13 to 77 years of age (median, 46). Plasma insulin, C peptide, proinsulin, and glucose concentrations were analyzed statistically in patients with benign and those with malignant insulinomas. Significant correlations were observed between plasma insulin and tumor size (r = 0.45; P = 0.001) and tumor volume (r = 0.41; P = 0.007) and between plasma C peptide and tumor size (r = 0.38; P = 0.004) and tumor volume (r = 0.33; P = 0.025). No differences between benign and malignant tumors were noted for plasma insulin, C peptide, glucose, nor tumor size or volume. No correlations were found between plasma insulin, C peptide, or glucose and number of tumors. The plasma levels of insulin and C peptide provide qualitative indications about the size of insulinomas.

Redistribution of nuclear antigens linked to cell proliferation and RNA processing in mouse oocytes and early embryos.

We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti-Sm and anti-RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2-cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its "speckles" co-localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti-Sm and anti-RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post-hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression.

Myosin and Actin from Aortae of Spontaneously Hypertensive and Normotensive Rats

Contractile proteins were extracted from thoracic aortae of 9-month-old spontaneously hypertensive stroke-prone (SHRSP) and normotensive Wistar Kyoto rats (WKY). The average wet weight of the aortae from SHRSP was approximately 1.6-fold heavier than that of WKY. On polyacrylamide gel electrophoresis of the myofibrillar extracts prepared from normalized, equal weights of aortae of SHRSP and WKY, both the compositions and concentrations of major polypeptides including myosin heavy chains and actin were nearly identical in these extracts. The myosins purified from these extracts contained identical light chains of about 20- and 17 kDa. Proteolytic peptide maps of the heavy chains of myosin from SHRSP were also indistinguishable from those of WKY, suggesting that the same isoforms of myosin are expressed in both aortae. From these results it is suggested that the qualitative differences may be small, if any, between the major contractile proteins in the aortae of SHRSP and those of WKY.

Prothymosin α and parathymosin: mRNA and polypeptide levels in rodent tissues

Blot hybridization analyses have established the presence of mRNAs for prothymosin α (ProTα) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProTα and its mRNA were found in highest concentrations in spleen and thymus, followed by lung, kidney, and brain, with lowest concentrations in liver. On the other hand, liver contained highest concentrations of ParaT and the mRNA for ParaT, with lowest levels present in spleen and thymus. In comparison to tissues from young (6–8 week) mice, older (18 month) mice contained lower concentrations (20–40%) of both polypeptides, with qualitatively similar decreases in mRNA content.

Lipopolysaccharide-mediated induction of RNA polymerase I activity and amount in murine B lymphocytes.

The effect of lipopolysaccharide on RNA polymerase I activity in primary cultures of murine B lymphocytes has been examined. In cells treated with mitogen for 48 h, the activity of RNA polymerase I was approximately 15 times greater than in control cells. In situ localization of RNA polymerase I using indirect immunofluorescence indicated that there was at least a 10-fold increase in the amount of this enzyme associated with nucleoli of 48 h mitogen-treated cells relative to control cells. Immunoblotting experiments demonstrated a similar increase in the concentration of the 190-kDa subunit bound to DNA; the concentrations of the other polymerase I-associated polypeptides did not correlate with rRNA synthesis. Assuming 1 mol of the 190-kDa polypeptide/mol of polymerase I, it was estimated that 2,300 and 30,000 molecules of enzyme were associated with rDNA in the unstimulated and stimulated B cell, respectively. Thus, an increased cellular concentration of the 190-kDa subunit of RNA polymerase I and its association with ribosomal DNA may be a crucial step in rRNA synthesis.