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At least 19 recordsLinked to original sources
PubMed1980

Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of subsites for donor nucleotide triphosphates.

tRNA nucleotidyltransferase incorporates both AMP and CMP into tRNA acceptors. Studies of the effects of nucleoside triphosphates, nucleotide analogues, and affinity reagents on AMP and CMP incorporation indicate that these residues are donated from different subsites. However, neither of these sites is completely specific for nucleoside triphosphate binding, and CMP can actually be incorporated from the AMP-donating site, although at a slow rate. The two donor subsites interact with each other, such that binding of a ligand to the ATP site stimulates incorporation from the CMP-donating site. This interaction accounts for the biphasic CTP saturation curve and the unusual effects of nucleoside triphosphates on CMP incorporation observed earlier. In addition to donating CMP, the CTP subsite also serves as the position of binding of the terminal C residue of tRNA-C-C and, in the absence of CTP, for binding of the terminal residue of tRNA-C. These results, together with those in the accompanying paper, have defined multiple accepting and donating subsites within the active site of tRNA nucleotidyltransferase, as predicted from our previous model for enzyme action (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468). However, since we have been unable to obtain definitive evidence for two CMP-donating sites, we have considered a modification of this earlier model which utilizes only a single CMP-donating site. Using these models, we discuss how the specificity of the donor and acceptor subsites ensures the accurate synthesis of the -C-C-A sequence of tRNA.

Adenosine Monophosphate↗
PubMed1994

Receptor-stimulated guanine-nucleotide-triphosphate binding to guanine-nucleotide-binding regulatory proteins. Nucleotide exchange and beta-subunit-mediated phosphotransfer reactions.

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[gamma-thio]triphosphate, GTP[S], and guanosine 5'-[beta, gamma-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein beta subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formyl-peptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine-nucleotide-binding regulatory proteins and suggest that, in addition to the nucleotide-exchange reaction, a (thio)phosphate-group-transfer process via guanine-nucleotide-binding regulatory protein beta subunits is involved in the receptor-stimulated binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins.

Binding, Competitive↗
PubMed2001

Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates.

The effects of extracellular nucleotide triphosphates on the stimulation of mucin production by airway epithelial cells were examined. The order of potency in stimulating mucin secretion in primary cultures of human tracheobronchial epithelial cells is: uridine 5'-triphosphate (UTP) approximately equal to adenosine 5'-triphosphate (ATP) approximately equal to ATP-gamma-S > uridine 5'-diphosphate approximately equal to adenosine 5'-diphosphate > alpha,beta-methylene ATP >> adenosine. However, only UTP can increase mucin gene (MUC5AC, MUC5B) expression; ATP and other analogues have no stimulatory effect. The stimulation of MUC5AC and MUC5B expression by UTP is time- and dose-dependent. A similar effect on the elevation of mucous cell population in mouse airway epithelium can be demonstrated in vivo by an intratracheal instillation of UTP-saline solution. The stimulatory effect of UTP or ATP on mucin secretion was inhibited by pertussis toxin, U73122, and Calphostin C, but not by PD98059, suggesting a G-protein/ phospholipase (PL) C/protein kinase (PK) C-dependent and mitogen-activated protein kinase (MAPK)-independent signaling pathway. However, the stimulatory effect of UTP on mucin gene expression was sensitive to pertussis toxin and PD98059, but not to Calphostin C and U73122, suggesting a G-protein/MAPK-dependent and PLC/PKC-independent signaling pathway. These findings are the first demonstration that UTP, a pyrimidine nucleotide triphosphate, can enhance both mucin secretion and mucin gene expression through different signaling pathways.

Adenosine Triphosphate↗
PubMed1983

Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide.

Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(II) catalyze hydroxyl free radical formation from H2O2. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of OH produced from H2O2 as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from H2O2 over the low level obtained in the buffer system only. The cations, Mg2+ and Ca2+, even at much higher than physiological levels and much higher than the level of added Fe(II), did not cause a substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H2O2 to yield OH. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated OH formation from H2O2 demonstrated that Fe(II) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(II) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing OH formation from H2O2; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H2O2 catalysis to yield OH. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when H2O2 is present with ferrous-nucleotide complexes.

Adenosine Diphosphate↗
PubMed1984

The Ca2+ uptake and the hydrolysis of various nucleotide triphosphates by human platelet membranes.

Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40-80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent = 15 microM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP.

Biological Transport, Active↗
PubMed2000

Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli.

Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [gamma-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [gamma-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.

Adenosine Triphosphatases↗
PubMed1999

Binding of nucleotide triphosphates to cardiotoxin analogue II from the Taiwan cobra venom (Naja naja atra). Elucidation of the structural interactions in the dATP-cardiotoxin analogue ii complex.

Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na+,K+-ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP.CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the N- and C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na+,K+-ATPase is also proposed.

Circular Dichroism↗
PubMed1980

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates.

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.

Adenosine Triphosphate↗
PubMed1981

Invited review: the role of nucleotide triphosphate in actin and tubulin assembly and function.

Both actin and tubulin, the major proteins of the cytoskeleton, bind nucleotide triphosphate (NTP) and exhibit the phenomenon of "polymerization-coupled" NTP hydrolysis. In this report I review the nature of polymerization-coupled NTP hydrolysis, and its possible role in the cellular function of actin and tubulin. Polymerization-coupled hydrolysis may be viewed as simply reflecting differences in the NTPase activity of free subunit as compared to polymer. Making assumptions concerning the values of various rate constants, it is possible to write expressions for the effects of NTP hydrolysis on the kinetics of polymerization. The role of NTP hydrolysis may be viewed in at least three different ways: 1) Hydrolysis alters the kinetics of assembly and disassembly. This leads to a consideration of the role of subunit flow in microtubule and microfilament function. 2) Hydrolysis is an essentially irreversible step that separates the assembly and disassembly reactions. This suggests a role of NTP in the regulation of polymer content during cellular cycles of assembly and disassembly. 3) NTP may allow transient stabilization of intersubunit bonds. This suggests a role of NTP in nucleation and possible regulation of nonequilibrium states of assembly.

Actins↗
PubMed2000

Influence of temperature on the dynamics of ATP, ADP and non-adenylic triphosphate nucleotides in vegetative and floral peach buds during dormancy.

The nucleotides test of endodormancy, which is based on the capacity of tissues to synthesize ATP and non-adenylic triphosphate nucleotides (NTP), cannot be used for floral buds, and it is of questionable use for vegetative buds. In an attempt to find an alternative test, we examined whether the dormancy state of vegetative and floral buds of trees exposed to different temperature conditions during the rest period is directly related to their ATP, ADP and NTP concentrations and ATP/ADP ratio. Once the buds had entered endo- or paradormancy, the nucleotide concentrations and the ATP/ADP ratio were low in the vegetative primordia and very low in the floral primordia. Only after the action of chilling, when the buds were considered to have completed the endodormancy and paradormancy phases, did the nucleotide concentrations increase, accompanied by a steep rise in ATP/ADP ratio. We conclude that the ATP/ADP ratio could be used to characterize the bud dormancy state by comparison with critical values of 1.5 for vegetative primordia and 1.0 for floral primordia.

Journal Article↗
PubMed1996

Hepatic nucleotide triphosphate regeneration after hypothermic reperfusion in the pig model: an in vitro P-NMR study.

The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.

2,3-Diphosphoglycerate↗
PubMed1994

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice.

The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCP-microtubules. It was possible to study GMPCPP-microtubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the half-time for GMPCPP hydrolysis from > 10 h to approximately 10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s-1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.

Animals↗
PubMed1999

Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis.

BACKGROUND: The transport of macromolecules between the nucleus and cytoplasm is an energy-dependent process. Substrates are translocated across the nuclear envelope through nuclear pore complexes (NPCs). Translocation requires nucleocytoplasmic transport receptors of the importin beta family, which interact both with the NPC and, either directly or via an adaptor, with the transport substrate. Although certain receptors have recently been shown to cross the NPC in an energy-independent manner, translocation of substrate-receptor complexes through the NPC has generally been regarded as an energy-requiring step. RESULTS: We describe an in vitro system that is based on permeabilised cells and supports nuclear export mediated by leucine-rich nuclear export signals. In this system, export is dependent on exogenous CRM1/Exportin1 - a nuclear export receptor - the GTPase Ran and nucleotide triphosphates (NTPs), and is further stimulated by Ran-binding protein 1 (RanBP1) and nuclear transport factor 2 (NTF2). Unexpectedly, non-hydrolysable NTP analogues completely satisfy the NTP requirements for a single-round of CRM1-mediated translocation of protein substrates across the NPC. Similarly, single transportin-mediated nuclear protein import events are shown not to require hydrolysable NTPs and to occur in the absence of the Ran GTPase. CONCLUSIONS: Our data show that, contrary to expectation and prior conclusions, the translocation of substrate-receptor complexes across the NPC in either direction occurs in the absence of NTP hydrolysis and is thus energy independent. The energy needed to drive substrate transport against a concentration gradient is supplied at the step of receptor recycling in the cytoplasm.

Adenosine Diphosphate↗
PubMed1994

Two distinct mechanisms for the translocation of proteins across the thylakoid membrane, one requiring the presence of a stromal protein factor and nucleotide triphosphates.

Cytosolically synthesized thylakoid lumen proteins are normally transported across the thylakoid membrane of chloroplasts as intermediate forms following cleavage of the full precursor polypeptides by a stromal peptidase. We have examined the mechanisms by which two lumenal proteins, the 33- and 23-kDa components of the oxygen-evolving complex, are translocated across the thylakoid membrane using an in vitro import assay and synthetic engineered intermediate forms of the two proteins as substrates. Efficient transport of the 23-kDa protein is observed in both the presence and absence of stromal extracts, but efficient import of the 33-kDa protein into thylakoids is completely dependent on the presence of stroma. The required stromal factor is heat-sensitive, macromolecular, and trypsin-sensitive. These findings thus represent the first evidence for the active participation of one or more stromal proteins in thylakoidal protein transport. We also show that import into thylakoids of the 33-kDa protein, but not of the 23-kDa protein, is absolutely dependent on the presence of nucleotide triphosphates. The data therefore indicate that at least two distinct mechanisms exist for the translocation of lumenal proteins across the thylakoid membrane.

Adenosine Triphosphate↗
PubMed1982

Reversed-phase ion-pair high-performance liquid chromatographic assay of 5-fluorouracil, 5'-deoxy-5-fluorouridine, their nucleosides, mono-, di-, and triphosphate nucleotides with a mixture of quaternary ammonium ions.

Several quaternary ammonium ions were evaluated in the development of a reversed-phase ion-pair high-performance liquid chromatographic assay for the separation of the known nucleosides and nucleotides of 5-fluorouracil (FU) and its analogue, 5'-deoxy-5-fluorouridine (5'-dFUR). The capacity factors of FU, 5'-dRUR, and their eight anabolites including 5-5-fluorouridjine and its mono-, di-, triphosphate and diphosphoglucose, 5-fluorodeoxyuridine and its mono;- and diphosphate nucleotides, were dependent on the chain length and concentration of the counter ions, pH, type of buffer, as well as the type of bonded stationary phase. Separation of FU, 5'-dFUR, their nucleosides and monophosphate nucleotides was readily achieved using tetrabutylammonium ion alone as the counter ion. However, under these conditions, the di- and triphosphate nucleotides were eluted from the column only after lengthy gradient elution and with poor reproducibility. Optimal conditions for a simultaneous separation of the ten fore-mentioned compounds were achieved using a two-step elution with a mixture of tetraethylammonium (C8) and tetrabutylammonium (C16) ions. The first eluent consisted of C8 and C16 ions in a mixture of acetate--phosphate buffer and methanol, the second eluent contained an additional 30 mM phosphate. FU, 5'-dFUR, their nucleosides and monophosphates and diphosphoglucose were separated by isocratic elution from a microBondapak C18 reversed-phase column using the first eluent; and the di- and triphosphate nucleotides were subsequently eluted, isocratically with the second eluent. This assay does not require gradient elution and can be completed within 50 min with good reproducibility.

Chromatography, High Pressure Liquid↗
PubMed1982

Assembly of microtubule protein: role of guanosine di- and triphosphate nucleotides.

A quantitative analysis of the interplay between guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) in microtubule assembly and accompanying GTP hydrolysis has been performed when tubulin was polymerized in the presence of microtubule-associated proteins (MAPs) which display an interfering GTPase activity. The use of adenylyl beta-imidodiphosphate, which specifically inhibits the MAPs GTPase activity, and of vinblastine (or podophyllotoxin), which specifically inhibits GTP hydrolysis due to tubulin, made possible a study of the extensive GTP hydrolysis associated to microtubule assembly. The results indicate that GDP binds to microtubule ends with an affinity comparable to GTP, thus strongly inhibiting both the elongation process and the steady-state GTP hydrolysis at microtubule ends. GDP shifts the equilibrium between tubulin and microtubules toward disassembly. The MAPs which are released from the microtubules during the GDP-driven depolymerization cluster on the remaining microtubules. The resulting increased stability of microtubules is quantitatively consistent with the decrease in the critical concentration of the polymerizing species GTP-tubulin.

Animals↗
PubMed1978

Kinetic analysis of cooperativity in tubulin polymerization in the presence of guanosine di- or triphosphate nucleotides.

In vitro polymerization of pig brain tubulin, highly purified and deprived of microtubule-associated proteins, was followed by turbidimetry. Treatment of the data yielded the relation existing between the observed turbidity and the amount of polymer formed. This allowed a kinetic analysis, according to Oosawa's theories, of the polymerization process, which consisted of a slow spontaneous nucleation followed by the growth process. The apparent elongation rate constant was closely related to the nucleation process and exhibited a highly cooperative variation with tubulin concentration. The cooperativity was indicative of the size of the nucleus which appears to remain the same whether sheets or microtubules are formed. Magnesium ions appear to play a role in the polymorphism of tubulin polymers, the proportion of microtubules to sheets increasing with magnesium ion concentration. From kinetic experiments evidence was provided for GDP binding in competition with GTP, with a sixfold lower affinity. The tubulin-GDP complex could participate in microtubules elongation, but was not able to form nuclei. The critical concentration of tubulin in the presence of GDP was roughly twice as high as in the presence of GTP.

Animals↗