Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.     

The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase.

Purification of aldehyde oxidoreductase from C. thermoaceticum, the first detected enzyme able to reduce reversibly non-activated carboxylic acids to the corresponding aldehydes (White, H., Strobl, G., Feicht, R. & Simon, H. (1989) Eur. J. Biochem. 184, 89-96), results in the generation of multiple forms of the enzyme. The specific activities for the viologen-mediated dehydrogenation of butyraldehyde for the two main forms of the purification procedure are 530 and 450 U/mg. Two forms of the enzyme composed of alpha,beta- and alpha,beta,gamma-subunits, can be differentiated. The latter binds to red-Sepharose and can be eluted very specifically with NADPH. In contrast to the alpha,beta-types the trimeric forms also catalyse the reversible reduction of oxidised viologen with NADPH (VAPOR activity). The dimer alpha,beta can oligomerize and the alpha,beta,gamma-trimer can easily form various oligomers or split off the gamma-subunit. The apparent molecular masses of the subunits alpha,beta and gamma are 64, 14 and 43 kDa. The alpha,beta-form reveals an apparent molecular mass of 86 kDa containing about 29 iron, 25 acid-labile sulphur, 0.8 tungsten and forms about 1 mol pterine-6-carboxylic acid by permanganate oxidation. The corresponding values of the trimer showing a mass of 300 kDa, are about 82 Fe, 54 S, 3.4 W and 2.5 pterine-6-carboxylic acid. In addition, 1.7 mol of FAD could be found which seems to be a component of the gamma-subunit. The aldehyde oxidoreductase from C. thermoaceticum and that from C. formicoaceticum (White, H., Feicht, R., Huber, C., Lottspeich, F. & Simon, H. (1991) Biol. Chem. Hoppe-Seyler 372, 999-1005) show qualitative similarities as far as the Fe, S, W and pterin content and the broad substrate specificity are concerned. However, there are also surprisingly marked differences with respect to composition and amino-acid sequence.     

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.     

Tetanus toxin receptor Specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells

A subclone of rat pheochromocytoma cells expresses high affinity receptors for tetanus toxin on differentiation with NGF [Walton, K.M., Sandberg, K., Rogers, T.B. and Schnaar, R.L. (1988) J. Biol. Chem. 263, 2055–2063]. In the presence of protein cross-linking agents, [¹²⁵I]tetanus toxin, bound to these cells at 0°C, forms a cross-linked product with apparent molecular weight of 120 kDa. The formation of [¹²⁵I]tetanus toxin conjugate involves the heavy chain of the toxin, is prevented by cold toxin and it is largely reduced by pretreating cells with proteases, The cross-linked product is formed only upon incubation of the toxin with NGF-differentiated cells. These results suggest that a protein with apparent molecular weight of 20 kDa is involved in the neurospecific binding of tetanus toxin.     

Conversion of endoglycoceramidase-activator II by trypsin to the 27.9 kDa polypeptide possessing full activity: purification of activator for endoglycoceramidase by trypsin treatment followed by trypsin-inhibitor agarose column application.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9 kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.     

Protein kinases of the thylakoid membrane

The claim of Racker and co-workers (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156 and Lucero, H. A., Lin, Z. F., and Racker, E. (1982) J. Biol. Chem. 257, 12157-12160) that two protein kinases, designated CPK1 (25 kDa) and CPK2 (38 kDa), are present in spinach thylakoid membranes was investigated in light of results from this laboratory (Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385) showing that 75-80% of the measurable protein kinase activity of isolated thylakoids is attributable to a protein kinase of 64 kDa apparent molecular mass. Extraction of thylakoid membranes with octyl glucoside/cholate according to the procedure of Lin et al. (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156) released proteins assignable to CPK1 and CPK2 on the basis of photoaffinity labeling with 8-azido-[32P]ATP. The 64-kDa protein kinase was present in this extract and accounted for greater than 80% of the total phosphotransferase activity toward lysine-rich histone as substrate; it was not labeled by the photoaffinity reagent. The three presumptive kinases were purified by ammonium sulfate precipitation, sucrose density gradient centrifugation, hydroxylapatite chromatography, and affinity chromatography. CPK1 was specifically eluted from Cibacron blue-Sepharose by 10 mM ATP; it electrophoresed on denaturing polyacrylamide gels as a single band with apparent molecular mass of 25 kDa. Its specific activity toward lysine-rich histone as substrate was approximately 250 pmol of phosphate transferred (mg protein)-1 min-1. The 64-kDa protein kinase was eluted from the affinity column by 1% (w/v) lithium dodecyl sulfate or from a histone IIIs-Sepharose affinity column by 0.25 M NaCl. Its specific activity towards lysine-rich histone was 100-200 times greater than that of CPK1. CPK2 eluted from the Cibacron blue affinity column in 10 mM NADP+; it had an apparent molecular mass of 38 kDa, possessed NADPH-dependent diaphorase activity (specific activity: 225 nmol of ferricyanide reduced (mg protein)-1 min-1), and cross-reacted with immunoglobulin raised against purified ferredoxin:NADP+ oxidoreductase, with which it was thus identified. Kinase activity was not detectable in CPK2 or in reductase isolated by conventional procedures.     

Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.     

Purification and characterization of the erythropoietin-sensitive membrane phosphoprotein, pp43.

We have shown previously that purified human erythropoietin rapidly alters the phosphorylation of an integral erythroid membrane protein, pp43 (Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936). We have now purified pp43 to apparent homogeneity and have prepared antibodies to it. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer of membrane proteins to nitrocellulose, the antibodies identified pp43 and a series of higher molecular weight antigenically related proteins, up to 50 kDa, in erythropoietin-responsive Rauscher murine erythroleukemia cells and in normal murine erythroid cells. Examination of purified subcellular fractions confirmed the localization of pp43 and the related proteins to the plasma membrane. Phosphorylation with [gamma-32P]ATP demonstrated that, in contrast to pp43, these higher molecular weight proteins were not phosphorylated. Marked differences in both the abundance of pp43 and related proteins and the degree of erythropoietin-sensitive pp43 phosphorylation were found between the plasma membranes of Rauscher cells and those of "non-responsive" Friend murine erythroleukemia cells. In addition only trace amounts of a 50-kDa antigenically related protein and no phosphorylated pp43 were detected in the plasma membranes of two erythropoietin-insensitive human erythroid cells lines, K562 and HEL. The results suggest that the abundance and degree of phosphorylation of pp43 and the antigenically related proteins is strongly correlated with the erythropoietin responsiveness of the particular erythroid cell types.     

Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation. Evidence for catalytically induced interchange of the subunits

Previously we have found that histone H1 and H3 of tsBN2 cells showing premature chromosome condensation (PCC) at nonpermissive temperature (40.5 degrees C) were phosphorylated extensively as in mitotic cells (Ajiro, K., Nishimoto, T., and Takahashi, T. (1983) J. Biol. Chem. 258, 4534-4538). Under the influence of various chemicals, both the prevention of the PCC induction and the suppression of H3 phosphorylation occurred simultaneously, whereas H1 phosphorylation did not. At the minimum concentration for the inhibition of PCC induction, H1 phosphorylation remained at the control level, but H3 phosphorylation was completely suppressed. Tryptic peptide analysis revealed that the H3 phosphopeptide in PCC was single, and it was observed in the same position as in mitosis. The results suggest that specific site(s) of H3 phosphorylation related to the maintenance of a condensed state of chromatin.     

The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump.

Dimerization (oligomerization) of the plasma membrane Ca2+ pump increases its activity (Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). Fluorescence titration on preparations of the purified eosin-labeled human erythrocyte ATPase has been used to monitor the oligomerization process. Calmodulin inhibits oligomerization, although it can bind to the oligomerized enzyme. Synthetic peptides corresponding to the calmodulin-binding domain of the pump stimulate its ATPase activity, indicating the formation of heterooligomers of the peptides with the pump. The oligomerization is prevented by the preincubation of the ATPase with calmodulin. Polyclonal antibodies against the synthetic calmodulin-binding domain inhibit its basal and its calmodulin-stimulated ATPase activity and prevent the formation of the oligomers. ATPase preparations truncated at the COOH terminus with calpain to a fragment of 124 kDa which does not contain the calmodulin-binding domain fail to oligomerize with the intact ATPase. The results show that the calmodulin-binding domain mediates the oligomerization of the Ca2+ pump.     

Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres.

Ryals et al. (Ryals, P.E., Pak, Y., and Thompson, G. A., Jr., (1991) J. Biol. Chem. 266, 15048-15053) have described and partially characterized phosphatidylinositol glycans (PI glycans) present in Tetrahymena mimbres. We now describe the time course of PI glycan labeling from exogenous [3H]myristate, [14C]glucosamine, and [3H]ethanolamine. Over the first 2-12 h following pulse radioisotope addition a sizeable proportion of the radioactivity associated with the protein pellet remaining after cell delipidation existed as PI glycans. These compounds were distributed throughout the cell, with the largest proportion at 12 h being associated with a fraction containing mitochondria, lysosomes, and peroxisomes. However, by 24 h radioactivity had nearly disappeared from the PI glycans and had become associated with proteins by a process that was almost totally inhibited by cycloheximide or tunicamycin. PI glycans appeared to be incorporated mainly into a protein migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a relatively broad band with an apparent molecular mass of 24-29 kDa. The exact mobility of the protein band within this molecular weight range was dependent upon the growth temperature of the cells. The apparent molecular masses of the principal PI-anchored proteins formed by other closely related Tetrahymena species varied widely, ranging from 22 to 76 kDa. The PI-anchored proteins may belong to a group of surface proteins known as immobilization antigens. Treatment of 24-h-labeled T. mimbres cells with phosphatidylinositol-specific phospholipase C in vivo released labeled proteins from the cells. Some labeled proteins were present even in the medium of control, non-phospholipase-treated cells. Tetrahymena PI glycans appear to accumulate in a metabolic pool from which they are gradually removed for attachment to externally oriented PI-anchored proteins. Tetrahymena is a versatile system well suited for studying the regulation of PI-anchored protein biochemistry.     

Structure and synthesis of intracellular proteoglycan in HL-60 human leukemic promyelocytes

The structure, biosynthesis, and metabolism of proteoglycans in the HL-60 human promyelocytes were studied by metabolic labeling in culture with [35S]sulfate, [3H]glucosamine, [3H]serine, and [3H]leucine. These cells synthesize a single predominant species of intracellular proteoglycan with an approximate molecular weight of 100,000. The cells contain about 1 microgram of proteoglycan/million cells. The proteoglycan is turned over within the cells in two apparent pools with half-lives of about 0.6 and 27 h, respectively. The fast pool represents secretion into medium in an apparently intact form, whereas the slow pool represents intracellular degradation to free chondroitin sulfate chains and smaller fragments. The proteoglycan contains a protein core with an apparent Mr on gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of about 20,000-30,000. To the core protein are attached an average of six or seven chondroitin sulfate chains, each with an Mr of about 10,000. The chondroitin sulfate chains contain approximately 85% 4-sulfated and approximately 15% nonsulfated disaccharides. The chondroitin sulfate attachment region of the core protein is essentially resistant to trypsin and elastase, whereas the remainder of the protein core is readily degraded by proteases. The size of the chondroitin sulfate attachment region peptide generated by trypsin was estimated to be approximately 5 kDa. Based on the molecular size, distribution of amino acids, protease susceptibility, and the extent of O-glycosylation, we propose that the intracellular proteoglycan characterized in this study is the translation product of a proteoglycan gene reported to be present in these cells (Stevens, R.L., Avraham, S., Gartner, M.C., Bruns, G.A., Austen, K.E., and Weis, J.H. (1988) J. Biol. Chem. 263, 7287-7291).     

Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues

At least five distinct carrier proteins form the family of mammalian cationic amino acid transporters (CATs). We have cloned a cDNA containing the complete coding region of human CAT-3. hCAT-3 is glycosylated and localized to the plasma membrane. Transport studies in Xenopus laevis oocytes revealed that hCAT-3 is selective for cationic L-amino acids and exhibits a maximal transport activity similar to other CAT proteins. The apparent substrate affinity and sensitivity to trans-stimulation of hCAT-3 resembles most closely hCAT-2B. This is in contrast to rat and murine CAT-3 proteins that have been reported to display a very low activity and to be inhibited by neutral and anionic L-amino acids as well as D-arginine (Hosokawa, H., et al. (1997) J. Biol. Chem. 272, 8717-8722; Ito, K., and Groudine, M. (1997) J. Biol. Chem. 272, 26780-26786). Also, in adult rat and mouse, CAT-3 has been found exclusively in central neurons. Human CAT-3 expression is not restricted to the brain, in fact, by far the highest expression was found in thymus. Also in other peripheral tissues, hCAT-3 expression was equal to or higher than in most brain regions, suggesting that hCAT-3 is not a neuron-specific transporter.     

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.     

Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A.

Phosphorylation of Bcl2 at serine 70 is required for its potent anti-apoptotic function. We have recently shown that Bcl2 phosphorylation is a dynamic process that involves the protein kinase C alpha and protein phosphatase 2A (PP2A) (Ruvolo, P. P., Deng, X., Carr, B. K., and May, W. S. (1998) J. Biol. Chem. 273, 25436-25442; and Deng, X., Ito, T., Carr, B. K., Mumby, M. C., and May, W. S. (1998) J. Biol. Chem. 273, 34157-34163). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2-dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain-of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC50 > 50 microM). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC50 = 14 microM). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.     

Apolipoprotein A-I induces translocation of protein kinase C[alpha] to a cytosolic lipid-protein particle in astrocytes

Apolipoprotein A-I (apoA-I) induces the translocation of newly synthesized cholesterol as well as caveolin-1 to the cytosolic lipid-protein particle (CLPP) fraction in astrocytes before its appearance in high density lipoprotein generated in the medium (Ito, J., Y. Nagayasu, K. Kato, R. Sato, and S. Yokoyama. 2002. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes. J. Biol. Chem. 277: 7929-7935). We here report the association of signal-related molecules with CLPP. ApoA-I induces rapid translocation of protein kinase Calpha to the CLPP fraction and its phosphorylation in astrocytes. ApoA-I also induces the translocation of phospholipase Cgamma to CLPP. Diacylglyceride (DG) production is increased by apoA-I in the cells, with a maximum at 5 min after the stimulation, and the increase takes place also in the CLPP fraction. An inhibitor of receptor-coupled phospholipase C, U73122, inhibited all the apoA-I-induced events, such as DG production, cholesterol translocation to the cytosol, release of cholesterol, and translocation of protein kinase Calpha into the CLPP fraction. CLPP may thus be involved in the apoA-I-initiated signal transduction in astrocytes that is related to intracellular cholesterol trafficking for the generation of high density lipoprotein in the brain.     

Control of pyrimidine biosynthesis in human lymphocytes. Inhibitory effect of guanine and guanosine on induction of enzymes for pyrimidine biosynthesis de novo in phytohemagglutinin-stimulated lymphocytes.

Human peripheral lymphocytes were incubated with Phaseolus vulgaris phytohemagglutinin. The induction of glutamine-utilizing carbamyl phosphate synthetase (EC 2.7.2.5) and aspartate transcarbamylase (EC 2.1.3.2) for pyrimidine biosynthesis de novo and the induction of uridine kinase were observed as described previously (Ito, K., and Uchino, H. (1971) J. Biol. Chem. 246, 4060-4065; Ito, K., and Uchino, H. (1973) J. Biol. Chem. 248, 389-392; Lucas, Z.J. (1967) Science 156, 1237-1240). By the addition of 1 mM guanine to the culture, the induction of the former two enzymes was inhibited, while that of uridine kinase was not, and even accelerated. An increase in the rate of [14C] bicarbonate incorporation into the acid-soluble uridine nucleotides via the de novo pathway for pyrimidine biosynthesis after phytohemagglutinin stimulation was inhibited by guanine, the incorporation rate being almost at the level of the control culture without phytohemagglutinin. Guanosine had a similar effect on pyrimidine biosynthesis. The induction of the three enzymes mentioned above was completely inhibited by adenine (1 mM). Guanine and guanosine seem to have a unique inhibitory effect on the induction of glutamine-utilizing carbamyl phosphate synthetase and aspartate transcarbamylase.