The peptidyl-puromycin reaction with Neurospora crassa ribosomes

Summary The reaction of the peptidyl-tRNA in an in vitro system from Neurospora crassa with puromycin has been studied by different experimental approaches. Ribosomes precharged with labelled polyphenylalanine have been prepared and the release of radioactivity, occurring after the reaction with puromycin, has been followed on sucrose density gradients. Furthermore the reaction of endogenous peptidyl-tRNA carried by ribosomes isolated from actively growing mycelia with ³H-puromycin has been characterized. By this latter technique it has been possible to evaluate the percentage of ribosomes engaged in protein synthesis in ribosomal populations isolated from mycelia in different stages of growth.Abbreviations used TF-1 aminoacyl transferase I - TF-2 aminoacyl transferase II - PM puromycin - ³H-PM puromycin-methoxy-³H dihydrochloride - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - TCA trichloroacetic acid - PEP phosphoenolpyruvate - tRNA transfer RNA - rRNA ribosomal RNA - PPO 2,5-diphenyloxazole - POPOP 1,4-bis-(5-phenyloxazol-2-yl) benzene     

Kinetic studies of peptide bond formation. Effect of chloramphenicol

There might be an undetermined order in the interaction of the substrates with the ribosomes in the reaction of CACCA-Leu-Ac with puromycin to form Ac-Leu-puromycin and CACCA (fragment reaction). Km 0°=6×10^-4 M for the puromycin · ribosome interaction. Chloramphenicol totally blocks the fragment reaction as a consequence of a single interaction with the ribosome of Kd 0°=2.2×10^-6 M. The inhibition by chloramphenicol of the fragment reaction is mixed competitive for puromycin.     

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 g/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C¹⁴ leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely under the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 g/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C¹⁴ leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed.     

Testing with puromycin for amino acyl trnas that limit the rate of peptide chain extension

Summary With puromycin one can recognize when the synthesis of a given protein is dependent on amino acyl tRNA that is present in rate limiting amount. We demonstrate this use of puromycin by its interaction with another inhibitor, L-o-methylthreonine. L-o-methylthreonine lowers the Ile-tRNA concentration in the cell, thereby inhibiting synthesis of proteins containing isoleucine. In certain rabbits, the a hemoglobin chain has three isoleucyl residues and the chain none. L-o-methylthreonine thus inhibits globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting a globin synthesis. Hence, puromycin is a more effective inhibitor when the Ile-tRNA concentration is lowered. Cycloheximide and sodium fluoride have different modes of action from puromycin. Neither synergizes with L-o-methylthreonine; instead, the interaction is less than additive. We have found that chain synthesis in rabbit reticulocytes is more sensitive than to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of chain elongation. The switch from fetal to adult hemoglobin in humans does not involve changes in limiting amino acyl tRNA because, for cord blood from infants of different developmental ages, the puromycin sensitivity of incorporation into and chains remains constant.     

Die hemmung der Phytochrom-induzierten Anthocyansynthese durch puromycin und 2-thiouracil

Zusammenfassung Die durch Phytochrom 730 bewirkte Anthocyansynthese des Senfkeimlings kann durch Puromycin und 2-Thiouracil gehemmt werden. Im Gegensatz zu Actinomycin D (Lange u Mohr, 1965) hemmen diese Substanzen die Anthocyansynthese auch dann fast völlig, wenn sie erst längere Zeit, z. B. 12 Std. nach Lichtbeginn appliziert werden. Der Effekt von 2-Thiouracil kann aufgehoben werden, wenn man die Keimlinge in ein Medium mit Uracil oder Wasser bringt. Auch die gleichzeitige Applikation von Uracil und 2-Thiouracil führt zu einer zumindest partiellen Aufhebung des Hemmeffektes von 2-Thiouracil. Aus den Kinetiken kann man folgern, daß die Lebensdauer des kurzlebigsten Enzyms, das an der Anthocyansynthese beteiligt ist, in der Größenordnung von 6 Std liegt. Der Schluß erscheint aufgrund der Abb. 1. u. 2 berechtigt, daß die Synthese dieses Enzyms über P₇₃₀ reguliert wird. — Die Wirkung von 2-Thiouracil kann in unserem Fall offensichtlich nicht nur damit gedeutet werden daß 2-Thiouracil in RNS engebaut wird und dadurch falsche RNS entsteht. Man muß vielmehr annehmen, daß das 2-Thiouracil auch eine direkte, kompetitive Enzymhemmung verursacht.

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The inhibitory effect of puromycin and 2-thiouracil on the phytochrome-mediated synthesis of anthocyanin

Summary The phytochrome-mediated anthocyanin synthesis of the mustard seelding, Sinapis alba L., can be stopped by the application of relatively low concentrations of puromycin and 2-thiouracil. While actinomycin D, which has been investigated in a previous paper (Lange and Mohr, 1965), will block phytochrome-induced anthocyanin synthesis only if it is applied before or at the onset of light, puromycin and 2-thiouracil will stop anthocyanin synthesis even if they are applied at a later stage, e.g., 12 hours after the onset of light. — The effect of 2-thiouracil can be reversed if the seedlings are transferred to a medium containing uracil or to blank water. The simultaneous application of 2-thiouracil and uracil also leads to at least a partial reversal of the 2-thiouracil effect. — The kinetics of the puromycin inhibition (Fig. 2) indicate that the enzyme with the shortest life-time among those enzymes which are involved in anthocyanin production has a life-time in the order of 6 hours. One may reasonably conclude on the basis of Fig. 1 and 2 that synthesis of this particular enzyme is controlled by phytochrome 730. — The effect of 2-thiouracil (Fig. 3) cannot — in our case at least — be understood by only assuming that 2-thiouracil will be incorporated into RNA and thus will lead to the formation of wrong RNA. Inhibition by 2-thiouracil is much faster than inhibition by puromycin (Fig. 2,3). We have rather to conclude that 2-thiouracil may exert its effect mainly through a direct competitive enzyme inhibition.

     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [³H]Serine incorporation was maximally inhibited by 3 × 10^?4m puromycin but unaffected by 10 ^?5m puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

The puromycin reaction mediated by yeast ribosomes in high salt

The extend of the reaction between puromycin and yeast peptidyl-tRNA prelabeled in vitro was determined by measuring the distribution of trichloroacetic acid precipitable material in isokinetic sucrose gradients in the presence of 0.5 M KCl.Thus it was found that increasing amounts of puromycin remove increasing amounts of peptidyl-tRNA from the 80S position in the gradient. The extend of the reaction, however, was independent of pretreatment of the ribosomes with inhibitors of the translocation indicating that peptidyl-tRNA at the donor and at the acceptor site of the ribosomes are equally accessible to puromycin at 0.5 M KCl.The exposure of both ribosomal binding sites to puromycin in high salt is accompanied by an enhanced reactivity of puromycin towards peptidyl-tRNA. The ED₅₀ determined by measuring the inhibition by puromycin of the poly-U dependent phenylalanine incorporation drops from 5×10^-5 M below 250 mM KCl to 5×10^-6 M at 300 mM and higher concentrations of KCl.     

Metabolic fate of initiation factors after inhibition of protein synthesis in Escherichia coli

Summary The metabolic fate of translation initiation factor after inhibition of protein synthesis by different means has been investigated. We have found a decay in initiation factor activity when protein synthesis is blocked by chloramphenicol but not during arginine starvation of PA1 (Rel^–) or PA2 (Rel⁺) strains or during puromycin incubation. These results suggest that inactivation of certain initiation factors occurs when the regeneration of ribosomal subunits from polysomes is inhibited in the cells.Complementation experiments indicate that IF₃ factor activity is preferentially affected during chloramphenicol treatment.Same preferential inhibition of IF₃ activity seems to occur during in vitro incubation of crude IF. 70S ribosomes or 30S subunits protect this factor against the inactivation. Preliminary results seem tosuggest that ATP is implicated in this in vitro inactivation process.     

Protein synthesis and wall extensibility in the Avena coleoptile

Summary The inhibitors cycloheximide and puromycin have been used to examine the relationship between protein synthesis and wall extensibility, as measured with an Instron, in Avena coleoptile segments. Cycloheximide at 4 g/ml almost totally inhibits both auxin-induced cell elongation and protein synthesis with only a slight lag. Wall extensibility is unaffected by the inhibitor if auxin is absent. If added prior to auxin, cycloheximide prevents auxin-induced wall loosening while if added after auxin it causes a substantial decline in the wall extensibility. With puromycin there is a 2–4 hr lag before growth and wall loosening are inhibited. These results support the conclusions that the proteins needed for wall loosening are unstable, and that continued protein synthesis is necessary to maintain the wall loosening process.     

CD4CD8 thymocytes are induced to cell death by a small dose of puromycin via ER stress

When the CD4⁺CD8⁺ thymic lymphoma cells were treated with puromycin, we found that most of the cells died at 0.3-1 μg/ml of puromycin within 24 h. However, cell death was greatly reduced when the dose of puromycin was increased. Similar dose-pattern of cell death was observed in thymocytes and the sensitivity to puromycin was greater in CD4⁺CD8⁺ thymocytes than CD4⁺CD8⁻ thymocytes. The induction of apoptosis was blocked by the protein synthesis inhibitor cycloheximide, and to some extent by transfection of Bcl-xL or Bcl-2 genes. Expression of GRP78 was up-regulated after treatment with a small dose of puromycin, and the cell death by puromycin was blocked in the presence of caspase 12 inhibitor. These results indicated that the induction of cell death by low-dose puromycin was due to endoplasmic reticulum stress. Furthermore, we found that dexamethasone, a synthetic glucocorticoid, and puromycin worked synergistically to induce cell death in thymocytes.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Elektronenmikroskopische und histoautoradiographische Befunde zur Puromycinwirkung auf ausreifende Plexus chorioideus-Zellen in vitro

Zusammenfassung Der Einfluß von Puromycin auf die Inkorporierung von Leucin-³H wird an kultivierten Plexus chorioideus-Zellen 9 Tage alter Hühnerembryonen untersucht. Die Kulturen zeigen unter Einwirkung von Puromycin eine deutliche Hemmung der Aufnahme von Leucin-³H. Die Aufnahme der markierten Substanz ins Cytoplasma wird im Vergleich zu Kontrolluntersuchungen prozentual mehr gehemmt als die Aufnahme des Leucins in den Kern.Unmittelbar nach dem Zusatz von Puromycin zum Nährmedium treten in den kultivierten Plexus chorioideus-Zellen Ansammlungen von multivesicular bodies und z. T. auch zahlreiche Lysosomen auf. Beide Organellen verschwinden im Verlauf der weiteren Kultivierung wieder fast vollständig aus den Zellen.Eine neuerliche Zugabe von markiertem Leucin nach der Puromycingabe zeigt, daß die beobachteten Wirkungen reversibel sind und daß die Puromycinwirkung nicht länger anhält, als diese Substanz im Nährmedium vorhanden ist.

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On the influence of puromycin on embryonic choroid plexus cells in vitro. An electronmicroscopic and histoautoradiographic study

Summary The influence of puromycin on the incorporation of leucin-³H into cells of choroid plexus of nine day old chicken embryos is studied.Puromycin causes a strong inhibition of the incorporation of leucin-³H into the cultures. Compared with controls, the inhibition of the uptake of the labelled substance is more pronounced in the cytoplasm than in the nucleus.Immediately after puromycin is added to the tissue culture medium an accumulation of multivesicular bodies and—to a certain degree—of lysosomes appears in the cells of the choroid plexus. Both organells disappear almost completely during the cultivation period.A second incubation with labelled leucin after puromycin shows that the observed effects are reversible and that puromycin acts only as long as it is present in the culture medium.The embryonic cells of choroid plexus develop normally even though treated with puromycin.

Die unreifen Plexus chorioideus-zellen entwickeln sich trotz der Puromycingaben normal.Das morphologische Differenzierungsergebnis wird nicht verändert.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

Inhibition of chitin biosynthesis in cultured imaginal discs: Effects of alpha-amanitin,actinomycin-D,cycloheximide, and puromycin

Summary Wing imaginal discs isolated from last instar larvae of the Indian meal moth,Plodia interpunctella, produced chitin when incubated in vitro with 2×10^–7 M 20-hydroxyecdysone. Chitin biosynthesis was initiated 8 h after the conclusion of a 24-h treatment with hormone. Simulataneous incubation of wing discs with 20-hydroxyecdysone and either inhibitors of RNA synthesis (alpha-amanitin, actinomycin-D) or inhibitors of protein systhesis (cycloheximide, puromycin) prevented chitin biosynthesis. We conclude from our results that RNA and protein synthesis must continue undiminished during the hormone-contact period, and that synthesis of protein, but not of new RNA is required during the posthormone culture period. Our findings are consistent with the hypothesis that ecdysteroids stimulate insect metamorphosis by promoting the synthesis of new RNA and protein during a hormone-dependent phase followed by hormone-independent protein synthesis.     

Reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC₅₀ of about 1 M. In the presence of brevetoxin the IC₅₀ increased to about 80 M. Reserpine inhibited binding of batrachotoxinin-A [³H]benzoate ([³H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 M, then a rebound stimulation from 0.1 to 0.8 M, followed by complete inhibition by 80 M. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC₅₀ of about 1 M. Reserpine at 1 M slightly reduced the off-rate of [³H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 M it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3–10 M elicited a partial inhibition of the binding of [³H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [³H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC₅₀ of about 2 M to an IC₅₀ of about 50 M. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.