THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

Colchicine-binding activity in particulate fractions of mouse brain

Both particulate and soluble fractions of brain homogenates bound [³H]colchicine. Approximately one-half of the total colchicine-binding activity in mouse brain was found in the particulate fraction. Of the particulate fractions, the microsomal and nerveending subfractions which sediment at the 1·0–1·2 m interface on sucrose gradients were richest in colchicine-binding activity. Intact microtubules were not found in these fractions, but colchicine-binding activity of these fractions may be related to the presence of microtubular protein.     

Colchicine-binding activity and neurotubule integrity in a rat sympathetic ganglion

The colchicine-binding activity of rat superior cervical ganglia was examined. Ganglia were cooled and re-warmed in the presence of either Cu²⁺ or of metabolic inhibitors. Electronmicroscopy showed that these treatments depolymerized the neurotubules. This depolymerization of neurotubules did not affect the colchicine-binding activity of ganglion homogenates but caused a two-fold increase in colchicine-binding by whole ganglia. This suggests that colchicine binds only to depolymerized neurotubule subunits and that colchicine-binding by whole ganglia can be used as a measure of polymerization of the neurotubule protein.The major part of the colchicine-binding activity of ganglion homogenates was found in the soluble fraction and was unstable. In the absence of divalent cations, 10⁻⁴ M vinblastine stabilised the soluble colchicine-binding activity.     

SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-³H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat γ-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from ¹⁴C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [¹⁴C]lysine plus [¹⁴C]glycine, [¹⁴C]histidine plus [¹⁴C]glutamate, [¹⁴C]glutamine alone and [¹⁴C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10–15min of incubation. Only 4–9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

Reduced permeability in CHO cells as a mechanism of resistance to colchicine

Colchicine resistant (CH^R) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [³H] colchicine uptake by whole cells and binding of [³H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CH^R lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

The occurrence of polysialogangliosides in a human trophoblast cell line

Several lines of evidence establish that the crystallizable aspartyl—tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl—tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [¹⁴C]glucose or [¹⁴C]glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein. Indeed immunoprecipitation of [¹⁴C]glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl—tRNA synthetase.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

The biosynthesis of glucagon in perfused rat pancreas

The biosynthesis of glucagon was studied by using the recirculated, isolated perfused rat pancreas. [³H]Tryptophan was initially incorporated into acid–ethanol-extractable protein, which on gel filtration was eluted with a molecular weight of about 9000 and contained a small amount of glucagon immunoreactivity. With longer incubation [³H]tryptophan incorporation into a second peak was obtained in an identical position with that of the majority of rat glucagon immunoreactivity. This peak of labelled protein exhibited migration characteristics on polyacrylamide-gel electrophoresis identical with those of rat glucagon and was identified as newly synthesized glucagon by demonstration of specific binding and dissociation behaviour with glucagon antibodies. The incorporation of [³H]tryptophan into acid–ethanol-extractable protein was inhibited by cycloheximide. High concentrations of glucose increased [³H]tryptophan incorporation into high-molecular-weight protein but decreased incorporation into proteins smaller than cytochrome c. The pattern of [³H]leucine incorporation into protein was similar to that of [³H]tryptophan.     

Bursicon-induced adenyl cyclase activity in the haemocytes of the American cockroach

Partially purified preparations of bursicon were injected, between ecdyses, into American cockroaches (Periplaneta americana) held at 4°C. After short periods of time, the haemocytes were removed and found to contain appreciable amounts of cyclic adenosine 5′-monophosphate (cAMP). Identical experiments using animals reared at 23°C did not result in the accumulation of cAMP. These and additional data suggest that phosphodiesterase is not active during the initial stages of bursicon-stimulated adenyl cyclase.Incubation of haemocyte soluble proteins in the presence of [³H]cAMP, followed by molecular sieve chromatography on P-60 polyacrylamide gel, shows the binding of cAMP to large molecular weight proteins. Similar experiments, but with phosphodiesterase inhibition revealed a reduced level of radioactive bound protein. From these studies one can assume that the haemocytes contain a soluble cAMP-dependent protein kinase.If either [¹⁴C]-leucine or [¹⁴C]-tyrosine is incubated with whole blood taken from animals between ecdyses, little or no uptake by the haemocytes can be seen. Identical studies, but with the addition of cAMP, results in a dramatic enhancement of amino acid uptake. It appears that bursicon stimulates blood-cell adenyl cyclase, which in turn produces cAMP. The cAMP subsequently activates a specific protein kinase that phosphorylates potential membrane proteins. The membrane phosphoprotein may be instrumental in the enhanced uptake of amino acids.     

Incorporation of L-tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine as single units into rat brain tubulin.

The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit.     

A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF₁) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkötter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [¹⁴C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [¹⁴C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [¹⁴]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [¹⁴C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [¹⁴C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [¹⁴C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [¹⁴C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

Specific affinity labelling of tubulin with bromocolchicine

Bromocolchicine, synthesized by substituting tho N-acetyl moiety of colchicine with a reactive bromoacetyl group, was found to be an affinity label for tubulin. Binding of [³H]colchicine to tubulin was competitively and irreversibly inhibited by bromocolchicine with a K_i value of 2.3 × 10^?5m. The affinity label could not be displaced by precipitating the protein with trichloroacetic acid and is thus covalently bound. Autoradiographs of brain high-speed supernatant proteins after their electrophoretic separation on sodium dodecyl sulphate/polyacrylamide gels showed that [³H]bromocolchicine reacted with four proteins, of which tubulin was one.Labelling of two of these proteins could be prevented by pretreatment of the brain extracts with α-bromoacetic acid, after which 70% of the covalently bound label was specifically located in the tubulin band. Up to 1.6 mol of affinity label could be bound per mol of tubulin, while under our experimental conditions 1 mol of protein bound irreversibly only 0.2 mol of [³H]colchicine. Autoradiography of sodium dodecyl sulphate/urea-polyacrylamide gels, which separate the subunits of tubulin, showed about 30% [³H] bromocolchicine bound to the α-subunit of tubulin and 70% to tho β-subunit.The irreversible binding site of colchicine was localized to the α-subunit, as labelling of only this subunit was inhibited by colchicine at high affinity label concentrations. At lower concentrations, colchicine inhibited the labelling of both subunits.Bromoacetic acid did not inhibit the reaction of the affinity label with the tubulin subunits, but increased the inhibition of [³H]bromocolchicine binding at lower concentrations of the affinity label in brain extracts preincubated with cold colchicine. This is interpreted to show a conformational change which takes place in the two subunits of tubulin upon binding of colchicine and results in the exposure of some of the binding sites of [³H]bromocolchicine to bromoacetic acid.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.