Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate

Natural and artificial manipulation of tissue nonspecific alkaline phosphatase activity indicates that pyrophosphate, phosphoethanolamine, and pyridoxal 5'-phosphate are among the natural substrates for this enzyme. Although inorganic phosphate has been recognized as a competitive inhibitor of this enzyme for many years, the influence of phosphate on alkaline phosphatase activity in serum under physiological conditions has not been previously reported. We examined the kinetics of tissue nonspecific alkaline phosphatase from bovine kidney and sera from 49 patients with a wide range of endogenous phosphate concentrations using pyridoxine 5'-phosphate as a substrate at pH 7.4. For the bovine kidney enzyme, the Km was 0.42 +/- 0.04 micromol/L, and the Ki for phosphate was 2.4 +/- 0.2 micromol/L. Analysis of the kinetics using pyridoxine 5'-phosphate in undiluted serum from 10 subjects with phosphorus ranging from 0.5-2.1 mmol/L and alkaline phosphatase activity ranging from 41-165 nmol/min x mL gave estimates for the Km of 56 +/- 11 micromol/L and for the Ki of 540 +/- 82 micromol/L for phosphate. This indicates that under physiological conditions alkaline phosphatase activity toward pyridoxine 5'-phosphate is reduced approximately 50% by the normal phosphate concentration and that it will increase or decrease significantly in response to changes in phosphate concentration within the ranges observed clinically.     

Differential effects of paclitaxel (Taxol) analogs modified at positions C-2, C-7, and C-3' on tubulin polymerization and polymer stabilization: identification of a hyperactive paclitaxel derivative

Our finding that an analog of paclitaxel (Taxol) modified at position C-2 (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substantially more active than paclitaxel in promoting tubulin assembly [Chaudhary et al. (1994) J. Am. Chem. Soc. 116, 4097-4098] led us to perform an analysis of the modulating effects of microtubule-associated proteins, GTP, and temperature on assembly and polymer stability. The analog always showed superior activity to paclitaxel in inducing polymerization where it fails to occur without drug, probably indicating a greater ability than paclitaxel to "hypernucleate" assembly. In contrast, much smaller differences in effects on polymer stability were observed. The analysis was extended to a large series of derivatives modified at positions C-2, C-7, C-10, and C-3', including docetaxel, a clinically important analog of paclitaxel. While analog stabilization of polymer was frequently observed, neither qualitative nor quantitative analysis of this property reliable predicted whether a compound would have enhanced hypernucleation activity relative to that of paclitaxel. Stabilization was often observed at substoichiometric analog concentrations, while even superstoichiometric concentrations of most compounds failed to induce extensive tubulin polymerization at low temperatures or in the absence of microtubule-associated proteins or GTP. Docetaxel was intermediate in activity between paclitaxel and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel in promoting assembly reactions. We conclude that the hypernucleation of tubulin assembly and polymer stabilization observed with paclitaxel represent two distinct properties of the drug. Our findings suggest that paclitaxel, docetaxel, and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel are able to interact with progressively smaller assemblages of tubulin at low temperatures or in the absence of microtubule-associated proteins or GTP.     

Changes in skeletal metabolism in pubertal girls can be revealed by biochemical parameters of calcium metabolism and bone turnover

Biochemical changes related to skeletal turnover in puberty were investigated in a sample of 67 girls aged 8-14 years. The following biochemical parameters were measured in serum: total calcium, phosphate, magnesium, total alkaline phosphatase, osteocalcin, and calcium and hydroxyproline in the second morning urine. Thirty-five premenarchal girls (8-11 years) had significantly lower serum calcium, and higher alkaline phosphatase and phosphate than those menstruating regularly (N = 32, 12-14 years). A statistically significant negative correlation of serum parameters and age was found for phosphate and alkaline phosphatase in all subjects, and for calcium and magnesium only in the premenarchal girls. These results indicated the more intensive processes of skeletal metabolism occurring in prepubertal age and early puberty to reflect in basic biochemical parameters of calcium and bone metabolism. Analysis of correlation between biochemical parameters showed alkaline phosphatase and phosphate to correlate positively with hydroxyproline excretion and negatively with urinary calcium in all subjects. In the subjects after menarche, osteocalcin correlated with alkaline phosphatase and phosphate. Thus, biochemical parameters indirectly reflected physiologic changes occurring with bone turnover in puberty. Variations in bone turnover during puberty, including a more pronounced bone formation during prepubertal or early stages, can be indirectly observed through biochemical parameters related to calcium and bone metabolism. Investigations of skeletal growth and puberty would benefit from specific markers of bone remodeling and "basic" biochemical parameters, as it might disclose subtle metabolic relationships.     

Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules

The interactions of nucleotides and their role in the polymerization of tubulin have been studied in detail. GTP promotes polymerization by binding to the exchangeable site (E site) of tubulin. The microtubules formed contain only GDP at the E site, indicating that hydrolysis of E site GTP occurs during or shortly after polymerization. Tubulin prepared by several cycles of polymerization and depolymerization will polymerize in the presence of ATP as well as GTP. Polymerization in ATP is preceded by a distinct lag period which is shorter at higher concentrations of ATP. As reported by others ATP will transphosphorylate bound GDP to GTP. Under polymerizing conditions the maximum level of GTP formation occurs at about the same time as the onset of polymerization, and the lag probably reflects the time necessary to transphosphorylate a critical concentration of tubulin. The transphosphorylated protein can be isolated and will polymerize without further addition of nucleotide. The transphosphorylated GTP is hydrolyzed and the phosphate released during polymerization. About 25% of the phosphate transferred from ATP is noncovalently bound to the subunit as inorganic phosphate and this fraction is also released during polymerization. The nonhydrolyzable analogue of GTP, GMPPNP, will promote microtubule assembly at high concentration. GMPPNP assembled microtubules do not depolymerize in Ca concentrations several fold greater than that which will completely depolymerize GTP assembled tubules; however, addition of Ca prior to inducing polymerization in GMPPNP prevents the formation of microtubules. Thus GTP hydrolysis appears to promote depolymerization rather than polymerization. GDP does not promote microtubule assembly but can inhibit GTP binding and GTP induced polymerization. GDP does not, however, induce the depolymerization of formed microtubules. These experiments demonstrate that tubulin polymerization can not be treated as a thermodynamically reversible process, but must involve one or more irreversible steps. Exchange experiments with [3H]GTP indicate that the "E" site on both microtubules and ring aggregates of tubulin is blocked and does not exchange rapidly. However, during polymerization and depolymerization induced by raising or lowering the temperature, respectively, all the E sites become transiently available and will exchange their nucleotide. This observation does not suggest a direct morphological transition between rings and microtubules. The presence of a blocked E site on the rings explains the apparent transphosphorylation and hydrolysis of "N" site nucleotide reported by others.     

Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.     

The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells

The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.     

Lactobacilli and vaginal host defense: activation of the human immunodeficiency virus type 1 long terminal repeat, cytokine production, and NF-kappaB

A mechanism-based screening program aimed at the discovery of new antimicrotubule agents from natural products yielded laulimalide and isolaulimalide, two compounds with paclitaxel-like microtubule-stabilizing activity. Treatment of A-10 cells with laulimalide resulted in a dose-dependent reorganization of the cellular microtubule network and the formation of microtubule bundles and abnormal mitotic spindles. Coincident with the microtubule changes, these two compounds induced nuclear convolution and the formation of multiple micronuclei. Laulimalide is a potent inhibitor of cellular proliferation with IC50 values in the low nanomolar range, whereas isolaulimalide is much less potent with IC50 values in the low micromolar range. In contrast to paclitaxel, both laulimalide and isolaulimalide inhibited the proliferation of SKVLB-1 cells, a P-glycoprotein overexpressing multidrug-resistant cell line, suggesting that they are poor substrates for transport by P-glycoprotein. Incubation of MDA-MB-435 cells with laulimalide resulted in mitotic arrest and activation of the caspase cascade of proteolytic enzymes that accompany apoptotic cell death. Laulimalide stimulated tubulin polymerization and, although less potent than paclitaxel, it was more effective. Laulimalide-induced tubulin polymers resembled paclitaxel-induced polymers, although the laulimalide-induced polymers appeared notably longer. Laulimalide and isolaulimalide represent a new class of microtubule-stabilizing agents with activities that may provide therapeutic utility.     

N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin: synthesis, chemical and enzymatic stability studies

Four N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin are prepared and characterized. Their enzymatic and chemical stability are assessed using high-performance liquid chromatography. The prodrug derivatives are shown to degrade stoichiometrically to Leu-enkephalin in phosphate buffer [t1/2 (0.05 M phosphate buffer without KCl): acetone prodrug (II) 930 min; cyclopentanone prodrug (III): 216 min; cyclohexanone prodrug (IV): 432 min; 4-methylcyclohexanone prodrug (V): 792 min]. Furthermore, the prodrugs are shown to afford global stabilization of the Leu-enkephalin molecule towards the enzymes, aminopeptidase N and angiotensin converting enzyme, primarily responsible for degradation of Leu-enkephalin at the blood-brain barrier and in plasma. Therefore, the 4-imidazolidinones, being metabolic stable and bioreversible, may be suitable prodrug candidates for delivery of Leu-enkephalin to important target areas such as the brain, if given intravenously.     

Serum calcium, phosphate and alkaline phosphate levels in epileptic children treated with phenobarbitone

A longitudinal study to estimate the serum calcium, phosphate and alkaline phosphatase levels of 89 ambulatory epileptic children, aged between 3 years and 12 years, and having generalised tonic-clonic seizures, was carried out. None was on any form of medication for the treatment of seizures prior to presentation. Each patient received only phenobarbitone during the period of study. Serum levels of the biochemical parameters were determined at presentation, 6 months and 12 months, while serum phenobarbitone levels were estimated at 6 months and 12 months. Mean serum calcium, phosphate and alkaline phosphatase of the patients remained within the normal range. Using the paired 't' test, the differences in the levels of the parameters at the three measurements were not statistically significant (P > 0.05). Serum phenobarbitone levels remained within the therapeutic range during the period of study. Our results show that over a 12-month period, serum levels of calcium, phosphate, and alkaline phosphatase, remain normal in ambulant epileptic children treated with phenobarbitone.     

Comparison of total and bone-specific alkaline phosphatase in patients with nonskeletal disorder or metabolic bone diseases

To evaluate the diagnostic validity of new assays for bone-specific alkaline phosphatase (BAP), we compared measurements of total alkaline phosphatase (TAP) in serum with results for three different assays of serum BAP in healthy adults (n = 119), patients with chronic nonskeletal disorders (n = 123), and patients with metabolic bone diseases (n = 113). Serum TAP was determined by a standard colorimetric assay, BAP by the methods of lectin precipitation (L-BAP), enzyme immunoassay (E-BAP), and immunoradiometric assay (I-BAP). Impairment of liver function resulted in significant increases of all alkaline phosphatase (AP) measurements, with the smallest changes being exhibited by E-BAP. Compared with the results by TAP, diagnostic sensitivity (i.e., of values exceeding the reference interval) was not improved by BAP, but receiver-operating characteristic (ROC) curve analyses revealed improved discrimination for primary hyperparathyroidism by E-BAP. These results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP. In most other clinical situations, serum TAP appears to provide sufficient clinical information; however, the cross-sectional study design used here allows no statement about the usefulness of BAP in serial measurements.     

The C terminus of beta-tubulin regulates vinblastine-induced tubulin polymerization

Oligoanions such as sodium triphosphate or GTP prevent and/or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastine-induced tubulin polymerization and abolishes the anion effect. Only the beta-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the alpha-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 microM block cleavage of the alpha-tubulin C terminus in the polymer, whereas cleavage of the beta-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pH-dependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastine-induced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the beta-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.     

Studies on human minor salivary gland secretions using the Periotron method

This assay is based on morphological changes of rat glioma cells treated with db-cAMP. The db-cAMP treatment induces a tubulin-dependent change causing the cells to acquire a spherical shape. Pretreatment with tubulin inhibitors brings about the disintegration of tubulin polymer and/or prevents its polymerization. Cells with inhibited tubulin fail to respond to db-cAMP treatment. Cells treated with inhibitors of tubulin polymerization are then separated from the spherical cells by aspiration. A semiautomated scanning procedure evaluates the final culture density and yields graphical data.     

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.     

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1

Cryptophycin 1 is a remarkably potent antiproliferative compound that shows excellent antitumor activity against mammary, colon, and pancreatic adenocarcinomas in mouse xenographs. At picomolar concentrations, cryptophycin 1 blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. The compound binds to tubulin, inhibits microtubule polymerization, and depolymerizes preformed microtubules in vitro. Its exceptionally powerful antitumor activity (many-fold greater than paclitaxel or the vinca alkaloids) raises important questions about its mechanism of action. By quantitative video microscopy, we examined the effects of cryptophycin 1 on the dynamics of individual microtubules assembled to steady state from bovine brain tubulin. At low nanomolar concentrations, in the absence of net microtubule depolymerization, cryptophycin 1 potently stabilized microtubule dynamics. It reduced the rate and extent of microtubule shortening and growing and increased the frequency of rescue. The results suggest that cryptophycin 1 exerts its antiproliferative and antimitotic activity by binding reversibly and with high affinity to the ends of microtubules, perhaps in the form of a tubulin-cryptophycin 1 complex, resulting in the most potent suppression of microtubule dynamics yet described.     

Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.     

Conformational studies of paclitaxel analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems

The conformations of two paclitaxel analogs modified at the C-2' position, 2'-deoxypaclitaxel and 2'-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2'-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2'-acetate, an analog in which the 2'-hydroxyl group hydrogen-bonding capacity has also been eliminated.     

Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin

Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.