Determination of myoglobin saturation of frozen specimens using a reflecting cryospectrophotometer.

This report describes a method and instrumentation for determining myoglobin (Mb) oxygen saturation in skeletal muscle. Canine gracilis is frozen in situ using a liquid N2-cooled copper block. Transverse section surfaces of frozen unstained muscle are observed at -110 degrees C using a microspectrophotometric system. The Mb saturation is determined using epi-illumination and a four-wavelength optical method. A special aperture permits illumination of a 20-microns-square area, and the radius of the catchment volume is estimated to be approximately 60 microns, with the strongest signal arising from the central region. The equibestic wavelengths used were 546.6, 570.5, and 584.1 nm. The method was validated using the nonlinear multicomponent analysis method of Lübbers. End-point (0 and 100% saturation) calibration was set using ischemic and adenosine-treated highly oxygenated muscles, respectively. The effects of hemoglobin (Hb) and metmyoglobin (metMb) signal contamination were evaluated experimentally and by computer-mixing simulations. Mb saturation determinations adjacent to large vessels are to be avoided. MetMb and capillary Hb do not interfere with the determination. The reproducibility of the method is estimated to be +/- 5%.

Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap.

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubule of 60 microns/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437-1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.

GTP hydrolysis during microtubule assembly.

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.

Structural features of human tracheobronchial mucus glycoprotein.

Electron microscopy of platinum-shadowed preparations of human tracheobronchial mucins showed very flexible filamentous structures that frequently occurred in an intricate random-coiled pattern of filament(s) surrounding a dense core-like domain. The filament(s) associated with cores accounted for 70-80% of the mass of the mucin preparation, the remainder being accounted for by free filaments. On aggregation, the molecules formed a large interwoven network quite different from the massive rope-like structures characteristic of sheep submaxillary mucin aggregates [Rose, Voter, Sage, Brown & Kaufman (1984) J. Biol. Chem. 259, 3167-3172]. Mild sonication resulted in extensive fragmentation of the tracheobronchial mucin molecules and yielded short filaments of various lengths, free cores and some cores associated with short filaments. Mucin glycopeptide fragments obtained by proteolytic digestion were flexible, core-free, filaments. The glycopeptides obtained by Pronase digestion were shorter than those obtained by tryptic digestion. The intricate structures of human tracheobronchial mucin differ markedly from the extended filaments reported for sheep submaxillary and human ovarian-cyst mucins but agree with the roughly spherical expanded model proposed for mucins by Creeth & Knight [(1967) Biochem. J. 105, 1135-1145] on the basis of hydrodynamic measurements.

The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism.

A model describing the nucleation and assembly of purified tubulin has been developed. The novel feature of this model is a two stage nucleation process to allow the explicit inclusion of the two-dimensional nature of the early stages of microtubule assembly. In actin assembly the small starting nucleus has only one site for subunit addition as the two-stranded helix is formed. In contrast, microtubule assembly begins with the formation of a small two-dimensional section of microtubule wall. The model we propose is a modification of the work of Wegner and Engel (Wegner, A., and Engel, J. (1975) Biophys. Chem. 3, 215-225) wherein we add a second stage of nucleation to directly account for lateral growth, i.e. the addition of a small number of subunits to the side of an existing sheet structure. Subsequent elongation of the sheets is treated in the usual way. The experimental system used to test this model was the Mg2+/glycerol induced assembly of purified tubulin. The computer simulation of the polymerization time courses gave a fairly good fit to experimental kinetics for our model, where the primary nucleus comprises two protofilaments, of four and three subunits, and lateral growth requires a three-subunit nucleus to initiate a new protofilament.

Effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein.

The structural features of native and deglycosylated ovine submaxillary mucin (OSM) were determined by electron microscopy of platinum unidirectionally shadowed preparations and by ultracentrifugation. Thin filamentous molecules, of which 90% were 100-230 nm in length with estimated diameters of 1.0-1.4 nm, were observed with dilute samples of OSM in high ionic strength solvents (5-30 micrograms/ml in 0.8 M NaCl or NH4Ac). Ultracentrifugation studies indicated that these filamentous structures were monomers and/or dimers. At higher mucin concentrations or in lower ionic strength solvents, OSM molecules were oligomers that appeared as long rope-like strands. Removal of sialic acid residues by incubation with Clostridium perfringens neuraminidase yielded filamentous structures similar to those observed with OSM and some smaller less extended structures. Subsequent removal of the GalNAc residues of asialo-OSM with C. perfringens alpha-N-acetylgalactosaminidase resulted in a dramatic change in appearance, from an extended filament to a globular form. The frictional ratios of OSM and deglycosylated OSM were consistent with the marked structural differences of these molecules. Native OSM had a frictional ratio of 3.09, comparable to that of highly asymmetric tropomyosin (3.22); deglycosylated OSM had a frictional ratio of 1.11, comparable to that of globular ovalbumin (1.08).

Tubulin rings: curved filaments with limited flexibility and two modes of association.

Tubulin rings have been previously identified as composed of linear polymers of tubulin subunits, equivalent to a protofilament in the microtubule wall but in a curved rather than a straight conformation. We have examined and measured a number of different ring structures obtained under different conditions. The preferred curvature is indicated by a single ring of 380 A outside diameter. Radially double rings consist of two coplanar rings of 460 A and 350 A outside diameter, held together by a pattern of eight identical contacts between the 40 A subunits in the inner and outer rings. In some circumstances a larger ring, 570 A diameter, can be added to the outside, or a smaller ring, 240 A diameter, may be added to the inside of the radially double ring, in both cases repeating the pattern of eight radial contacts. The distortion of the filament from its relaxed 380 A diameter curvature apparently can be made without disrupting the longitudinal bond between subunits in the filament, but must be stabilized by the energy of the radial contact. All of these rings (single and radially double and triple) are observed to associate axially to form pairs or in some cases larger stacks. The radially double rings or an axially associated pair of these (quadruple ring) may also associate to form crystals. These are thin plates, up to 100 micrometers in extent and several micrometers thick which have been of limited use so far in diffraction studies because of irregularities in the packing of adjacent rings.

Polycation-induced assembly of purified tubulin.

Several different polycations have been found that can substitute for the microtubule-associated proteins, or tau factor, in facilitating assembly of tubulin that has been purified by ion exchange chromatography. In low concentrations of the polycation diethylaminoethyl-dextran, 7 mg of tubulin is pelleted per 1 mg of polycation added. Under conditions favorable to microtubule assembly the entire pellet is seen by electron microscopy to consist of "double wall microtubules", which are essentially identical to normal microtubules in subunit structure and arrangement. When assembly is inhibited approximately the same amount of tubulin is pelleted, but it is in the form of clusters of curved sheets or filaments apparently related to tubulin rings. When conditions are changed to favor assembly, the tubulin within these clusters appears to reassemble to form the double wall microtubules.