Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.

Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.

Kinesin and kinesin-like proteins comprise a superfamily of microtubule-dependent molecular motors important for various functions in intracellular organelle movements, mitosis, meiosis, and karyogamy (for reviews see Goldstein (1993), Walker and Sheets (1993), and Bloom and Endow (1994)). Among the at least seven families and several (as yet) single representatives, the family of "conventional" kinesins is the best characterized. The founding member of this family was identified concurrently in chick brain (Brady, 1985) and squid neural tissue (Vale et al., 1985a). Motors closely related to squid kinesin have subsequently been isolated from various tissues and organisms including bovine brain (Bloom et al., 1988;Kuznetsov and Gelfand, 1986), sea urchin eggs (Scholey et al., 1985), Drosophila (Saxton et al., 1988), and bovine adrenal medulla (Murofushi et al., 1988). A gene closely related to conventional kinesin has been found in Caenorhabditis elegans (Patel et al., 1993). In addition, recent evidence suggests that conventional kinesin may exist in two or more isoforms, at least in mammals (Navone et al., 1992;Niclas et al., 1994). Conventional kinesins have several properties in common: (i) they can be purified from cell extracts in sufficient quantities for biochemical and biophysical studies (Vale et al., 1985a;Brady, 1985;Scholey et al., 1985); (ii) they are composed of two 110 -130-kDa heavy chains and two 55-85-kDa light chains (Bloom et al., 1988;Kuznetsov et al., 1988); (iii) the heavy chains possess a globular N-terminal motor domain where the ATP and microtubule binding sites are located, a central ␣-helical (coiled-coil) stalk, and a globular C-terminal domain where the light chains bind (Hirokawa et al., 1989;Yang et al., 1989;Gauger and Goldstein, 1993); (iv) both the heavy and light chains exhibit a high degree of sequence homology throughout their length; (v) finally, the members of this family have been strongly implicated as force-generating proteins for the transport of membrane-bound organelles (Dabora and Sheetz (1988), Schroer et al. (1988), ; for review see Walker and Sheetz (1993) and Bloom and Endow (1994)). Taken together these properties suggest that the conventional kinesins are a family of closely related proteins whose molecular and biochemical properties are remarkably highly conserved.
Recently, we have identified an organelle motor from a "lower" eukaryote, the filamentous fungus Neurospora crassa. A molecular phylogenetic analysis using the N-terminal motor domain suggests that this motor, termed Nkin, 1 is a distant relative of the family of conventional kinesins identified in animals. Nkin exhibits several unusual structural and functional properties, such as a high rate of microtubule transport, a lack of copurifying polypeptides, a second p-loop motif, and the presence of a highly conserved peptide motif in the C terminus. The first organelle motor related to conventional kinesins identified in fungi, Nkin represents an intriguing "outsider" of this otherwise homogeneous protein family. To link the biochemical and biophysical properties of these motors to their biological function, we have analyzed the enzymatic, biophysical, and motility properties of Nkin and compared them, where possible, with those of animal kinesins.

EXPERIMENTAL PROCEDURES
Cell Cultures-N. crassa wild type 74A was grown in Vogel's minimal medium at 25°C under continuous aeration and illumination with white light for 14 -16 h as described by Sebald et al. (1979).
Preparation of Tubulin-Microtubule protein from pork brain was prepared using three cycles of polymerization and depolymerization according to the method of Shelanski et al. (1973) with modifications according to Mandelkow et al. (1985). Tubulin was further purified by * This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 184 and a grant from the Friedrich-Baur-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: MCD Biology, University of Colorado, Boulder, CO 80309. phosphocellulose column chromatography according to the method of Weingarten et al. (1975) and stored at Ϫ80°C.
Purification of Kinesin from N. crassa-N. crassa was grown for 16 h, harvested, and ground with quartz sand (Merck) in the presence of AP100 buffer (100 mM PIPES, pH 6.9, 2 mM MgCl 2 , 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml p-tosyl-L-arginine methyl ester, 10 g/ml soybean trypsin inhibitor, 1 g/ml aprotinin, 1 g/ml pepstatin, 1 g/ml leupeptin). After a low speed centrifugation at 10,000 ϫ g for 20 min to remove the sand and cell debris, a high speed supernatant (S2) depleted of organelles was prepared (120,000 ϫ g for 1 h). Tubulin was polymerized with 1 mM GTP and 10% Me 2 SO (v/v), stabilized with taxol (0.7 M), and added to the S2 at a concentration of 0.7 mg/ml. Additional taxol (7 M) as well as apyrase (5 units/ml; Grade VIII, Sigma) were added, and the mixture was incubated on ice for 1 h. In other experiments 2-4 mM AMPPNP alone or 0.2 mM AMPPNP in combination with 5 units/ml apyrase were used instead. The microtubules were sedimented (100,000 ϫ g for 30 min), carefully resuspended in AP100/50 mM KCl, and centrifuged through a 10% sucrose cushion in AP100 (90,000 ϫ g for 30 min). The resulting microtubule pellet (P4) was resuspended in AP100 with 5-10 mM ATP and placed on ice for 10 min. After centrifugation (100,000 ϫ g for 30 min), the resulting supernatant (S5) was tested in a gliding assay (see below). The S5 was stored on ice and normally stayed active for several days. For additional purification the motor activity was fractionated on a linear 5-20% sucrose gradient (5 ml) in AP100 and centrifuged at 130,000 ϫ g in a Beckman SW 50.1 rotor for 13 h. Fractions of 100 -300 l were collected from the bottom and checked for motor activity in gliding assays. For gel filtration a superose-6 column was used in fast protein liquid chromatography (Pharmacia Biotech Inc.). The column was equilibrated with 3 volumes of AP100, 150 mM NaCl, 1 mM dithiothreitol, 0.05 mM ATP. 400 -500-l fractions were collected. Fractions shown to be active in a gliding assay were usually concentrated for further use (microcon tubes, Amicon).
Motility Assay-The motor activity of Nkin was measured using an in vitro motility assay (Vale et al., 1985b) and video-enhanced DIC microscopy (Allen et al., 1981) as described in Steinberg and Schliwa (1995). For each data point the velocity of at least 6 microtubules (slow velocities) or 15 microtubules (fast velocities) from at least two different Nkin isolations were determined. All assays were performed in AP100; no differences were observed between sucrose-gradient fractions and gel filtration fractions. Because the motility of the Neurospora kinesin was very sensitive to the absence of nucleotides, the motor was stabilized with 0.05 mM ATP or ATP␥S in the sucrose gradients and the column buffer, respectively. This nucleotide "contamination" had no significant effect on the motility as tested in several control experiments. To determine the K m for microtubule motility, the motor solution was placed in a flow-through chamber. After adsorption of Nkin the column buffer was replaced by fresh AP100, followed by microtubules and AP100 with the desired nucleotide concentration. To determine the K m for microtubule motility, the velocities at a range of ATP concentrations were plotted against the nucleotide concentration (three different preparations).
ATPase Assay-The ATPase activity of purified motor (gel filtration fractions) was measured with the Malachite Green method (Itaya and Ui (1966); modified from Lill et al. (1990)). Column-purified peak fractions of motor were concentrated to ϳ100 g/ml Ϫ1 using microcon tubes (Amicon) and incubated in a reaction mixture with MgATP in several concentrations and either taxol-stabilized microtubules (1 g/ml Ϫ1 ) or AP100 buffer alone. To reduce nucleotide contamination, the microtubules were polymerized at a high concentration (Ͼ10 mg/ml Ϫ1 tubulin) with just 0.1 mM GTP. After for 10 min at 30°C, the reaction was stopped by the addition of 0.34% Malachite Green, 1% ammonium molybdate, 1 M HCl,0, 1% Triton X-100. The appearance of malachitephosphate complex was measured at 640 nm and corrected for phosphate contamination in the microtubule and the ATP solution by subtracting a control value from an experiment containing all ingredients except the motor. The A 640 of each data point was plotted against the ATP concentration and the K m values of three different kinesin preparations determined from these plots.
Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (1970) using 7% polyacrylamide gels. Gels were stained with Coomassie Blue or silver nitrate according to Blum et al. (1987).
Estimation of Stokes Radius-The Stokes radius was determined using gel filtration chromatography (Andrews, 1970) on a superose-6 column (Pharmacia) in AP100, 150 mM NaCl, 0.05 mM ATP. The calibration proteins (Boehringer Mannheim) and their Stokes radii (from Andrews, 1970) were ferritin (5.9 nm), aldolase (4.5 nm), bovine serum albumin (3.55 nm), cytochrome c (1.64 nm), and ␤-galactosidase (6.9 nm). In three independent experiments the peak positions of the standards were determined using an UV detector. From these the average elution volumes (V e ) were calculated. The void volume (V 0 ) was determined using high molecular weight blue dextran. A calibration curve was obtained by plotting the Stokes radii of the standards versus V e /V 0 . In nine independent experiments the Stokes radius of Nkin was determined from V e /V 0 .
Estimation of the Sedimentation Coefficient-200 l of purified Neurospora kinesin (gel filtration fraction or S5) were fractionated on in a 5-20% sucrose gradient in AP100 supplemented with 1 mM phenylmethylsulfonyl fluoride and 0.05 mM ATP at 4°C for 13 h at 37,000 rpm (Beckman, SW 50.1 rotor). Following centrifugation, 70 -100-l fractions were collected from the 5-ml gradients and analyzed by SDSpolyacrylamide gel electrophoresis. The fractions containing Nkin were checked for motility. The elution volume of Nkin was determined in four independent experiments using freshly isolated motor. The same was done two times for the standard proteins catalase (11.30 S), aldolase (7.40 S), bovine serum albumin (4.22 S), and ovalbumin (3.55 S; Boehringer Mannheim). The elution volume of the standard proteins was plotted against their S w,20 values, and the resulting calibration curve was used to estimate the sedimentation coefficient of Nkin.
Calculation of Molecular Weight, Diffusion Coefficient, and Axial Ratio-The native molecular weight was calculated from the sedimentation coefficient and the Stokes radius using the Svedberg equation (Cantor and Schimmel, 1980), assuming a partial specific volume of (0.725 g Ϫ1 cm 3 ; Andrews, 1970;Cantor and Schimmel, 1980). The diffusion coefficient D w20 was calculated from the Stokes radius according to the equation D w20 ϭ K t/6 r stokes . The axial ratio of Nkin, assuming it is an ellipsoid, was estimated from the Perrin parameter using a polynomial calibration curve of Perrin parameters versus axial ratios of other proteins (Cantor and Schimmel, 1980).
Protein Determination-Protein concentrations were determined according to the method of Bradford (1976) using catalase as a standard.
Chemicals-All chemicals were obtained from Sigma unless otherwise indicated.

RESULTS AND DISCUSSION
Native Nkin was isolated from cytoplasmic extracts of hyphae based on its nucleotide-sensitive binding to and release from pig brain microtubules (Steinberg and Schliwa, 1995). A typical experiment yielded 10 -20 g of motor from 30 g of packed hyphae. We routinely used apyrase to deplete ATP from cytoplasmic extracts for microtubule binding, but motor could also be isolated using AMPPNP as originally described (Brady, 1985;Vale et al., 1985a). The best purification was achieved by FIG. 1. Isolation of Neurospora kinesin. An organelle-free cytoplasmic extract of Neurospora wild type cell (S2) was incubated for 1 h at 4°C with 0.8 mg/ml taxol-stabilized microtubules under ATP-depleting conditions and sedimented (P3). The pellet was resuspended in isolation buffer with 50 mM salt and centrifuged through a 10% sucrose cushion. The supernatant (S4) was discarded, and the microtubule pellet (P4) was resuspended in release buffer containing 10 mM MgATP. For further purification the kinesin-containing supernatant (S5) was run over a gel filtration column and highly purified Neurospora kinesin was obtained (NKIN). Microtubule gliding was observed in P3, P4, S5, and the column fraction. The proteins were analyzed in SDS-polyacrylamide gel electrophoresis on a 7% gel and stained with Coomassie Brilliant Blue. the combination of apyrase and a low amount (0.2 mM) of AMPPNP, followed by gel filtration over a superose-6 fast protein liquid chromatography column, which yielded protein fractions that are 85-90% pure Nkin by densitomety of Coomassiestained polyacrylamide gels (Fig. 1). In the absence of ATP and microtubules, the motor quickly lost its ability to promote microtubule motility. Therefore, care was taken to maintain a minimum concentration of 0.05 mM ATP or ATP␥S during density gradient centrifugation or gel filtration. As discussed in Steinberg and Schliwa (1995) purified Nkin consists of a polypeptide doublet at 105/108 kDa, where the higher molecular mass form most likely represents a phosporylated isoform whose proportion varied from preparation to preparation. Despite our efforts, co-purifying polypeptides could not be detected. Most of the experiments to be described here were carried out with Nkin purified by gel filtration and sucrose density gradient centrifugation. Some of the standard microtubule gliding assays were also performed with material released from microtubules with ATP, in which Nkin constitutes about 30% of the total protein. No differences were found in these experiments to those obtained with gel-filtered material.
Biophysical Properties of Nkin-The biophysical properties of Nkin in comparison with bovine brain kinesin are summarized in Table I. The apparent molecular mass of Nkin after gel filtration was determined to be ϳ490 kDa, which corresponds roughly to that of urease (Stokes radius, 6.4 nm). The Stokes radius of Nkin was 6.27 Ϯ 0.53 nm (n ϭ 9) (Fig. 2), and the diffusion coefficient was 3.41 ϫ 10 Ϫ7 cm 2 s Ϫ1 (n ϭ 9). Sucrose gradient centrifugation was used to determine the sedimentation coefficient (Fig. 3). Five independent measurements yielded a value of 8.8. Ϯ 0.16 ϫ 10 Ϫ13 sec Ϫ1 . Using the Svedberg equation, the native molecular mass of Nkin was calculated to be 227 kDa (range, 199 -260 kDa; n ϭ 9). Light chains or other co-purifying polypeptides could not be found even though Nkin was prepared under conditions that allow for the isolation of both heavy and light chains of other conventional kinesins. However, this does not eliminate the possibility that light chains were lost during preparation because the properties of proteases of fungi are not well known. The native enzyme therefore may differ from animal kinesins, which are heterotetramers of two heavy and two light chains. The issue of the presence or the absence of light chains is currently being addressed using polymerase chain reaction with degenerate primers and screening of a Neurospora cDNA library with an appropriate cDNA probe.
The fundamental hydrodynamic and diffusion properties of Nkin reported here are those of an elongated molecule. Its Stokes radius, however, is considerably smaller than that of bovine kinesin, suggesting that Nkin is more compact than other conventional kinesins. This is reflected also in a smaller axial ratio of 12:1, as opposed to bovine brain kinesin, whose axial ratio is 20:1 (Bloom et al., 1988) for the "folded" form of kinesin and more like 40:1 for the fully extended molecule (Hackney et al., 1992).
Motility Properties of Nkin-The motility characteristics of Nkin in comparison with other conventional kinesins are summarized in Table II. One of the most dramatic differences between Nkin and other kinesins existed in terms of its rate of [ ϫ 10 7 cm 2 s Ϫ1 ] 3,34 Ϯ 0,20 2.24 Ϯ 0.21 Axial ratio ϳ12 ϳ20 d a All estimations concerning gel filtration were done nine times; the S w20 coefficient was measured four times. b Bloom et al., 1988. c Estimated from Stokes radius or D w20 coefficient for globular proteins summarized in Andrews (1970). d Folded conformation.

FIG. 2. Stokes radius of Neurospora kinesin.
A series of globular proteins were separated by gel filtration and V e /V 0 was plotted against the Stokes radii of these calibration proteins (n ϭ 3; Andrews (1970)). The average V e /V 0 of the Neurospora motor (n ϭ 9) was determined, and its Stokes radius was calculated from the regression equation. BSA, bovine serum albumin; ␤-gal, ␤-galactosidase.

FIG. 3. Sedimentation coefficient of Neurospora kinesin.
Globular calibration proteins were separated on a 4.5-ml 5-20% sucrose density gradient (n ϭ 2). Fractions of ϳ70 l each were analyzed by SDS-polyacrylamide gel electrophoresis, and the elution volume for each protein was plotted against its sedimentation coefficient. The mean elution volume (n ϭ 5) was used to calculate the sedimentation coefficient of Nkin from the regression equation. BSA, bovine serum albumin. motility. Among the kinesin motors identified so far, it was clearly the fastest. At saturating levels of ATP (2-4 mM), the rate of microtubule movement in standard gliding assays was 2.6 Ϯ 0.5 m s Ϫ1 (range, 2.1-3.8 m s Ϫ1) . It is important to note that this rate of in vitro motility corresponds well with the rates of in vivo movement of organelles (Steinberg and Schliwa, 1993), which usually range from 1 to 4 m/s. Microtubule-dependent motors that produce microtubule gliding at 2 m/sec or more have been isolated from Dictyostelium discoideum (Mc-Caffrey and Vale, 1989), Acanthamoeba castelanii (Kachar et al., 1987), and another fungus, Syncephalastrum racemosum, 2 but their molecular identity has not been established. The minimum concentration of motor required was 30 -40 g/ml, and the pH optimum was between 6 and 9. The movement of beads with adsorbed motor molecules on stationary microtubule was slower, namely, 1.6 Ϯ 0.2 m s Ϫ1 , corresponding to ϳ55% the rate of microtubule gliding. Like bovine brain and sea urchin kinesin, Nkin produced microtubule gliding in the presence of other naturally occurring nucleoside triphosphates (Table II). However, in contrast to other conventional kinesins, Nkin was less effective in the use of these natural nucleoside triphosphates (Shimizu et al., 1991). Interestingly, no motility was seen with MgITP, which promoted microtubule gliding at substantial rates with sea urchin kinesin (Cohn, 1989). We have also used four different ATP analogues in a gliding assay with Nkin and find that three of them (2Ј-deoxy-ATP, 8-azido-ATP, and ATP␥S) elicited a response similar to mammalian kinesin. A major difference was found with etheno-ATP, which was used very effectively by Nkin but not bovine brain kinesin.
It was reported recently that microtubules polymerized in the presence of a nonhydrolyzable GTP analogue, GMPCPP, have a higher flexural rigidity (appear stiffer; Venier et al., 1994) and that they are transported at a 30% higher rate in gliding assays than GTP microtubules . When gliding assays were done with GMPCPP microtubules, such an increase in the rate of microtubule movement was not observed, suggesting that microtubule stiffness does not alter microtubule velocity in the case of Nkin.
MgATP-dependent microtubule gliding has been found to be inhibited by a variety of compounds. In contrast to other conventional kinesins, Nkin appeared to be much more sensitive to some of these inhibitors. Thus, MgADP, a competitive inhibitor (Cohn et al., 1989), reduced the rate of microtubule gliding to about 50% at a ratio of ϳ1:1 to MgATP, whereas a 4-fold excess was needed for sea urchin kinesin (Cohn et al., 1989). Nkin also was more sensitive toward AMPPNP. Both findings are consistent with a lower affinity of Nkin for ATP, reflected also in the K m values of both ATPase activity and microtubule motility (see below).
Microtubule gliding activity of Nkin was relatively insensitive to sodium orthovanadate and N-etylmaleimide. The mode of inhibition is unexpected. With increasing concentrations of 2 G. Steinberg and M. Schliwa, unpublished results.   Vale et al., 1985. b Porter et al., 1987. c Saxton et al., 1988. d Cohn et al., 1989. e Howard et al., 1989. f Shimizu et al., 1991 Range of velocities in several experiments (5-20 microtubules each) from at least two different isolations. Percentage relative to ATP in parenthesis. All nucleotides and derivates were added with equimolar Mg 2ϩ .
h Vale et al., 1994. i Values indicate the ratio at an inhibition velocity of ϳ50%. j Scholey et al., 1989 Gelfand, 1986. m Hackney, 1988. n Gilbert and Johnson, 1993. o Cohn et al., 1987. p Penningroth et al., 1987 both reagents, there was a progressive release of microtubules from the coverslip surface, whereas the rate of movement of microtubules seemed virtually unaffected. At concentrations of orthovanadate of Ͼ70 M and concentrations of N-etylmaleimide of Ͼ3-5 mM, virtually no microtubules were left attached to the coverslip. However, a microtubule occasionally contacting the surface may be transported for several micrometers at normal rates before detaching again. ATP produced microtubule gliding in a saturable manner. The velocity obeyed a generalized Michaelis-Menten-like law (Leibler and Huse, 1993), as demonstrated by linearity in double-reciprocal plots of motility rates versus ATP concentration (Lineweaver-Burk plot; Fig. 4). In independent experiments with three different preparations of Nkin, linear regression analysis yielded apparent K m values of 187, 340, and 393 M ATP.
Native Nkin possessed an ATPase activity that was stimulated approximately 6-fold by microtubules (Steinberg and Schliwa, 1995). As already discussed by Huang and Hackney (1994) and Steinberg and Schliwa (1995), the ATPase rates determined in these assays are inconsistent with the rates of motility observed, suggesting that the ATPase properties of kinesin isolated by conventional procedures do not reflect the properties of the molecule in vivo. Like microtubule motility, the ATPase activity obeyed Michaelis-Menten kinetics (Fig. 5), yielding K m values of 112, 145, and 194 M ATP in three indepentent experiments. The ratio of the K m for motility (average 150 M) and the K m for ATPase activity (average 306 M) was ϳ2ϫ (Leibler and Huse, 1993). Studies with sea urchin, Drosophila, and bovine brain kinesin have yielded K m values for microtubule gliding ranging from 10 to 60 M (Porter et al., 1987;Saxton et al., 1988;Cohn et al., 1989;Howard et al., 1989); K m values of ATPase activity have been determined to be 10 -20 M for bovine brain kinesin (Kuznetsov et al., 1986(Kuznetsov et al., , 1989 and 31 M for recombinant Drosophila kinesin (Gilbert and Johnson, 1993). Thus for both K m values, the values of Nkin are approximately an order of magnitude higher. Whether the high rate of microtubule movement is related to the low affinity for ATP remains to be determined. In a theoretical analysis, Leibler and Huse (1993) compared the motors myosin and kinesin and predicted the ratio of the K m for motility and the K m for ATPase activity to be approximately 10 for myosin and 1-2 for kinesin. Even though the K m values of Nkin differ markedly from those of other kinesins, the ratio of the Michaelis-like constants for velocity and ATPase activity is approximately 2, as in the case of other conventional kinesins. Thus the basic features of the cross-bridge cycle of Nkin probably are similar to those of other kinesins. Nkin therefore seems to be a molecular motor of the "porter" type (Leibler and Huse, 1993), in contrast to myosin and probably also dynein, FIG. 4. Relationship between microtubule gliding velocity and ATP concentration. Column-purified motor was attached to clean coverslips, 0.06 mg/ml taxol-stabilized microtubules were added, and the probe was sealed in a flow-through chamber. After rinsing the chamber with ATP-free buffer the desired MgATP concentration was added, and motility was recorded on a video tape. The measured velocities were plotted against the ATP concentration (top). The K m was calculated from a Lineweaver-Burk plot (bottom).
FIG. 5. Microtubule-stimulated ATPase activity of Neurospora kinesin. The ATPase activity was determined by measuring the change in the optical density at 640 nm caused by the complex of phosphomolybdate and Malachite Green (Itaya and Ui, 1965). The enzymatic activity was monitored in the presence of 1 mg/ml taxol stabilized microtubules and plotted against the ATP concentration (top). The K m was calculated from a Lineweaver-Burk plot (bottom). which belong to the "rower" class of molecular motors with a ratio of the K m values of Ͼ10. The distinction between these two classes of motors on the basis of their K m values for motility and ATPase activity is in agreement with recent studies that directly demonstrate that the chemomechanical cycles of kinesin and myosin are indeed fundamentally different (Romberg and Vale, 1993;Gilbert et al., 1995). It will be interesting to determine whether the high rate of motility of Nkin is based on a larger step size, a shorter cycle time, or both.