P22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm, Manduca sexta, was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 13700 (inhibitor A) and 8000 (inhibitor B) by Sephadex G-75 gel filtration. SDS-polyacrylamide gel electrophoresis under non-reducing conditions gave a molecular weight estimate of 15000 for inhibitor A and 8500 for inhibitor B. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8300 and 9100 for inhibitor A and inhibitor B, respectively, suggesting that inhibitor A is a dimer. Isoelectro-focusing on polyacrylamide gels focused inhibitor A as a single band with pI of 5.7, whereas inhibitor B was resolved into two components with pIs of 5.3 and 7.1. Both inhibitors A and B are stable at 100° C and at pH 1.0 for at least 30 minutes, but both are inactivated by dithiothreitol even at room temperature and non-denaturing conditions. Inhibitors A and B inhibit trypsin, chymotrypsin, plasmin, and thrombin but they do not inhibit elastase, papain, pepsin, subtilisin BPN' and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors A and B. Inhibitor A and inhibitor B form stable complexes with trypsin. Stoichiometric studies showed that inhibitor A combines with trypsin and chymotrypsin in a 1:1 molar ratio. The inhibition constants (Ki) for trypsin and chymotrypsin inhibition by inhibitor A were estimated to be 1.45 x 10-8 M and 1.7 x 10-8M, respectively. Inhibitor A in complex with chymotrypsin does not inhibit trypsin (and vice versa) suggesting that inhibitor A has a common binding site for trypsin and chymotrypsin. The amino terminal amino acid sequences of inhibitors A and B revealed that both these inhibitors are homologous to the bovine pancreatic trypsin inhibitor (Kunitz) . Quantitation of the trypsin inhibitory activity in the hemolymph of the larval and the pupal stages of Manduca sexta showed that the trypsin inhibitory activity decreased from larval to the pupal stage. Further, inhibitor A at the concentration tested caused approximately 50% reduction in the rate of proteolytic activation of prophenoloxidase in a hemocyte lysate preparation from Manduca sexta, suggesting that inhibitor A may be involved in the regulation of prophenoloxidase activation. However, inhibitor B was not effective even at three times the concentration of inhibitor A. Since activation of prophenoloxidase has been suggested to resemble the activation of alternative pathway of complement, the effect of inhibitors A and B and the hemolymph of Manduca sexta on human serum alternative pathway complement activity was evaluated. The results showed that, although inhibitors A and B do not affect human serum alternative complement pathway, other proteinaceous component(s) in Manduca sexta hemolymph interact(s) and cause(s) an inhibition of human serum alternative complement pathway when tested using rabbit erythrocyte hemolytic assay.

The photon receptor, rhodopsin, and the GTP-binding regulatory protein, transducin, belong to a family of G protein-coupled receptors. The activation process through which guanine nucleotide exchange of the G protein is accomplished was investigated utilizing these components of the visual transduction system. Rhodopsin, modelled as an enzyme in its interaction with substrates, transducin and guanine nucleotides, was characterized to catalyze the G protein's activation by a double-displacement mechanism. Remarkable allosteric behavior was observed in these kinetic studies. Equilibrium binding studies were performed to investigate the molecular basis of the positive cooperative behavior between transducin and rhodopsin. These experiments show that the origins of the allosterism must arise from oligomeric assemblies between receptor and G protein. The determined Hill coefficient, nH = 2, suggests that at least two transducin molecules are involved, and the Bmax parameter a1so indicates that multimeric assemblies of rhodopsin may participate in the positive cooperative interactiions. Physical studies of transducin in solution were performed and do not indicate the existence of a dimeric structure, in contrast to the kinetic and binding experiments which analyze interactions at the membrane surface. Since the latter environment represents the native surroundings in vivo, aspects of the allosteric behavior must be considered for a complete understanding of the signal transduction mechanism. The reported findings are interpreted in the context of homologies between other G protein-coupled receptor systems in order to develop a model for the molecular basis of the mechanism and regulation of this mode of signal transduction.

The Tec family tyrosine kinases Itk, Tec and Rlk are expressed in T cells. Previous studies have established that these kinases are critical for TCR signaling, leading to the activation of PLCγ1. To further understand the functions of Tec kinases in T cell activation, we took three different approaches. First, we performed a thorough analysis of CD28-mediated signaling events and functional responses with purified naïve T cells from Itk-/- mice and a highly controlled stimulation system. Data from this set of studies definitively demonstrate that CD28 costimulation functions efficiently in naïve CD4+ T cells in the absence of Itk. Second, in order to further study the functions of Tec kinases in vivo, we generated transgenic mouse lines expressing a kinase-dead (KD) mutant of Tec on the Itk-/-Rlk-/- background, hoping to study mice that are functionally deficient for all three Tec kinases. The results hint the importance of the Tec kinases in T cell development and/or survival. Finally, in order to identify potential transcriptional targets of Itk, we used microarray technology to compare global gene expression profiles of naïve and stimulated Itk-/- versus Itk+/- CD4+ T cells. This analysis provided a short list of differentially expressed genes in Itk-/- versus Itk+/- CD4 T cells, providing a starting point for further studies of Itk in T cell activation. Collectively, these studies clarified the role of Itk in CD28 signaling, revealed some unexpected aspects of Tec family kinases in T cells, and indicated potential targets of Itk-dependent signaling pathways in T cells.

Summary Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death. Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli. To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons. In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells.

In mammals a single receptor protein binds both insulin-like growth factor II (IGF-II) and mannose 6-phosphate (Man 6-P) containing ligands, most notably lysosomal enzymes. However, in chick embryo fibroblasts IGF-II binds predominantly to a type 1 IGF receptor, and no IGF-II/Man 6-P receptor has been identified in this species. In order to determine if chickens possess an IGF-II/Man 6-P receptor, an affinity resin (pentamannosyl 6-phosphate (PMP) Sepharose) was used to purify receptors from chicken membrane extracts by their ability to bind mannose 6-phosphate. Then 125I-IGF-II was used to evaluate their ability to bind IGF-II. These experiments demonstrate that nonmammalian Man 6-P receptors lack the ability to bind IGF-II, suggesting that the ability to bind IGF-II has been gained recently in evolution by the mammalian Man 6-P receptor. The second area of study involves the serum form of the IGF-II/Man 6-P receptor. This receptor had been detected in the serum of a number of mammalian species, yet its structure, function, regulation, and origin were unknown. Initial studies, done with Dr. R. G. MacDonald, showed that the serum receptor is truncated such that the C-terminal cytoplasmic domain of the cellular receptor is removed. These studies also demonstrate a regulation of serum receptor levels with age, similar to that seen for the cellular receptor, and that the serum form of the receptor existed in several forms which appeared intact under nonreducing conditions, but as multiple proteolytic products upon reduction. Finally, these studies demonstrated that both the cellular and serum IGF-II/Man 6-P receptors are capable of binding IGF-II and Man 6-P simultaneously. In studies on the serum form of the IGF-II/Man 6-P receptor that I have conducted independently, the regulation of the serum IGF-II/Man 6-P and transferrin receptors by insulin has been demonstrated. In these studies, insulin injected into rats subcutaneously resulted in a time and dose dependent increase in serum receptor levels. Finally, to investigate the relationship of the serum IGF- II/Man 6-P receptor to the cellular form of the receptor, pulse chase experiments were performed. These experiments demonstrate that the soluble (serum form released into the medium) receptor is a major degradation product of the cellular receptor. Furthermore, the lack of detectable amounts of the lower Mr soluble receptor intracellularly and the parallel relationship of cell surface and soluble receptor suggest that the proteolysis is occurring from the cell surface. Finally, a number of experiments suggest that the degradation rate depends upon the conformation state of the receptor: binding of IGF-II or Man 6-P makes the receptor more susceptible to proteolysis while the presence of lysosomal enzymes prevents receptor proteolysis. In summary, the serum form of the IGF-II receptor is a proteolytic product of the cellular form of the receptor. The rate of release depends upon the number of receptors at the cell surface and the binding state of the receptor. In circulation, the receptor retains the ability to bind both types of ligands, it thus may serve as an IGF binding protein and/or a lysosomal enzyme binding protein. These results suggest a model whereby the cellular receptor is proteolytically cleaved by a plasma membrane protease to produce a short membrane anchored fragment and the serum receptor. In vivo this pathway serves as the major degradative pathway of the IGF-II/Man 6-P receptor, with the serum form being cleared from circulation by further degradation and reuptake.

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo.

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.

In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes. The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase. The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes. The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.