Membrane-associated nucleoside diphosphate kinase from rat liver. Purification, characterization, and comparison with cytosolic enzyme

Previous studies from this laboratory have proposed that membrane-associated nucleoside diphosphate kinase (m-NDP kinase) may play a role in regulation of adenylate cyclase by channeling GTP, an essential cofactor of adenylate cyclase regulation, into GTP-binding protein (Gs) in a hormone-dependent manner. To understand the true role of m-NDP kinase, in the present study, the m-NDP kinase was solubilized and purified to apparent homogeneity from rat liver purified plasma membranes and characterized in comparison with the cytosolic enzyme purified from the same tissue (s-NDP kinase). Some physical properties determined were: molecular weight (monomer), 18,300; sedimentation coefficient (s20,w), 6.2 S; isoelectric point (pI), 6.0. These values and kinetic parameters of the m-NDP kinase were almost identical to those of the s-NDP kinase whose characteristics were more extensively studied. A peptide mapping study of the 125I-labeled m- and s-NDP kinases gave essentially identical patterns. Polyclonal antibodies against the s-NDP kinase, which also cross-reacted with the m-NDP kinase, were prepared. Immunoblotting studies with the affinity-purified antibodies revealed that the monomer molecular weight of the purified m- and s-NDP kinases was identical to the values of unpurified enzymes present in membranes and crude extract. These results demonstrate that the purified m-NDP kinase underwent no remarkable modification during solubilization and purification, and that the m- and s-NDP kinases are quite similar in protein structure, if at all different. The physiological relevance of the m-NDP kinase in relation to the adenylate cyclase system is discussed.     

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.     

Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.     

Characterization of the detergent solubilized receptor for gastrin-releasing peptide

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

The microtubule-associated nucleoside diphosphate kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.     

Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.     

A monomeric form of human erythrocyte membrane acetylcholinesterase

Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained.     

Release of adenylate cyclase stimulating, GTP-binding protein, Gs from human erythrocyte membranes under the effect of fluoride-ion and guanine nucleotides

Human erythrocyte membranes were incubated in the presence of sodium fluoride or guanylylimidodiphosphate (GppNHp), a nonhydrolysable GTP analog. After centrifugation at 100000 g the activity of the adenylate cyclase-stimulating GTP-binding protein, Gs, was detected in the supernatant fraction. The release of the Gs activity from the membranes closely resembles Gs activation by GppNHp. The Gs activity release from the GppNHp-induced membranes is characterized by a lag period. The nucleotide concentration causing a half-maximal solubilization is about 9.10(-7) M. Approximately 50% of the Gs activity released from sodium fluoride-treated human erythrocyte membranes was associated with the cytoskeletal fraction extracted by a low ionic strength solution. The data obtained suggest that Gs exists in the membrane at lease in two compartmentalized states and is solubilized from both states during its activation.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

Direct interaction between membrane-associated nucleoside diphosphate kinase and GTP-binding protein(Gs), and its regulation by hormones and guanine nucleotides

In previous studies we have proposed that the membrane-associated nucleoside diphosphate kinase (m-NDP kinase) may play a role as a GTP channeling machinery for adenylate cyclase regulation by hormones. In this study, whether the m-NDP kinase has a direct interaction with the component (GTP-binding protein (Gs)) of the glucagon- and beta-adrenergic agonist-sensitive adenylate cyclase systems in rat liver membranes was examined by extraction with octylglucoside, followed by immunoprecipitation by affinity-purified monospecific anti-NDP kinase antibodies. The results demonstrated that the m-NDP kinase and the Gs were extractable as a complexed form and that the complex formation was reversibly regulated, through cell surface receptors, by hormones which had an ability to cause activation of the rat liver adenylate cyclase. Also, it was suggested that guanine nucleotides rather than hormones were primary regulators of the m-NDP kinase-Gs interaction. These results were discussed in relation to the regulatory cycle of the Gs of adenylate cyclase system.     

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).     

Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases α and β. Interaction with rhodopsin—transducin complex and thermal stability

To elucidate the physicochemical basis of differences between the isoforms of mammalian multifunctional nucleoside diphosphate kinase (NDP), we investigated the recombinant rat homohexameric NDP kinases alpha and beta, consisting of highly homologous alpha or beta subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase alpha(1-130)beta(131-152) and NDP kinase beta(1-130)alpha(131-152)) and tagged derivatives (NDP kinase HA-alpha(1-130)beta(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta). The thermal stability of these proteins and the ability of some of them to interact with the rhodopsin-transducin (R*Gt) complex have been studied. It was found that NDP kinase alpha, NDP kinase alpha(1-130)beta(131-152), and NDP kinase HA-alpha(1-130)beta(131-152) were similar in their thermal stability (T(1/2) = 61-63 degrees C). NDP kinase beta, NDP kinase beta(1-130)alpha(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta were inactivated at a lower temperature (T(1/2) = 51-54 degrees C). NDP kinase HA-alpha(1-130)beta(131-152) interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta(1-130)alpha(131-152) and NDP kinase beta with the photoreceptor membranes under the same conditions was very weak. It is suggested that the variability of the region V1 is a structural basis for the multifunctionality of NDP kinase hexamers in the cell.     

The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.     

Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.     

Comparative Study of Recombinant Rat Nucleoside Diphosphate Kinases α and β By Intrinsic Protein Fluorescence

Abstract

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases a and β, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme β, NDP kinase α is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186–190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes α and β both contain the same three tryptophan (Trp78, 133, 1nd 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase a undergoes spectral changes in the pH range 5–7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase β under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme α resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes α and β in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes α and β may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase α: (i) rather long-wavelength emission of NDP kinase a at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).     

Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori

Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity.     

Tubulin-associated nucleoside diphosphokinase.

Microtubule protein, prepared by cycles of polymerisation and dissociation, contained a nucleoside diphosphokinase (NDP kinase) activity (EC 2.7.4.6). This activity was not intrinsic to the tubulin dimer or the so-called microtubule-associated proteins. The NDP kinase had the following properties. (1) The enzyme existed in a low-molecular-weight form and in association with the complex of microtubule-associated proteins and tubulin (i.e. multimeric tubulin). (2) The low-molecular-weight species was also formed by dissociation of multimeric tubulin by salt or by removal of microtubule-associated proteins on phosphocellulose. (3) GDP bound to the exchangeable site of multimeric tubulin and also GDP derived from the E site of the tubulin dimer was a substrate for the NDP kinase. (4) The NDP kinase showed a 7-fold increase in activity during ATP-dependent microtubule assembly. On the basis of these properties, it is proposed that microtubule protein contains an NDP kinase specifically associated with tubulin and its functions.     

Megakaryocyte colony growth-supporting activities in human plasma: modification by platelets and platelet membranes

Human megakaryocyte colonies are grown in methylcellulose with platelet-poor plasma and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) as a source of megakaryocyte colony stimulating factor (MEG-CSF). The megakaryocyte colony growth-supporting activity in human plasma can be absorbed by intact platelets or degranulated platelet membranes. It was possible to recover the activity by solubilizing platelet membranes with cholic acid. Filtration of the solubilized platelet membrane preparations through a Sephadex G-100 column yielded at least two activity peaks. The molecular weight of these two activities differs from that of the growth-promoting activity in PHA-LCM.     

Nucleosidediphosphate kinase associates with rings but not with assembled microtubules

Microtubules reassembled in vitro from chick brain contain significant nucleosidediphosphate (NDP) kinase activity (EC 2.7.4.6) although the specific activity decreases with successive cycles of reassembly. However, while the recovery of microtubule protein, as a function of initial protein concentration, exhibits a critical concentration below which there is no polymerisation, the recovery of NDP kinase activity is, at low initial protein concentrations, directly proportional to the initial protein content indicating that microtubule protein and the kinase activity are independently recovered. This was confirmed by pelleting the reassembled microtubules through a sucrose cushion: the specific activity of the pelleted microtubules was reduced by approximately 90%. By contrast, when cold-dissociated microtubule protein, which is predominantly in the form of rings, is fractionated on a Biogel A 15 m column the microtubule protein and NDP kinase activity coeluted in the void volume and the specific activity remained constant throughout the ring fraction. Similarly, when microtubules were dissociated in the presence of NDP kinase the enzyme bound to the generated rings. These results suggest that NDP kinase binds preferentially to the rings compared with the microtubules, and a model is proposed to account for the presence of this enzyme in pellets of microtubule protein.