Cytosolic phospholipase A2 shows burst kinetics consistent with the slow, reversible formation of a dead-end complex.

Cytosolic phospholipase A2 catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. In the present research, a "burst" of arachidonate which precedes a somewhat slower, linear rate (upsilon) of product formation was observed and characterized using covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate. The magnitude of the burst (pi) was enzyme dependent, in both the presence and absence of glycerol. Upon subsequent addition of enzyme after the primary burst was complete, a second burst of arachidonate production was observed. This is consistent with the effect resulting from an enzyme effect and not from changes in the substrate. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol as the carrier phospholipid instead of DMPM greatly reduced the rate of hydrolysis without a large effect on the pi/upsilon ratio, consistent with the burst not being the result of limitations in the lateral diffusion rate of phospholipids within the covesicles. When the assay is performed in the presence of glycerol, the burst phenomenon was also observed with the monoarachidonoyl glycerol transacylase product which shows that the effect occurs through a common mechanism. The burst and subsequent linear rate of hydrolysis are highly temperature dependent, with a pronounced increase in the pi/upsilon ratio as the temperature is increased from 35 to 45 degrees C. A mechanism in which a slow equilibrium between an active and less active (inactive) state of substrate-bound enzyme is proposed. This may provide a means by which the enzyme is switched off after a few hundred turnovers in order to prevent unabated phospholipid hydrolysis in cells which may be deleterious to membrane integrity.     

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).     

Proteolysis mediated by the membrane‐integrated ATP‐dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane‐integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40–60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water‐soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Toward mechanical manipulations of cell membranes and membrane proteins using an atomic force microscope

Recent advances in the use of the atomic force microscope (AFM) for manipulating cell membranes and membrane proteins are reviewed. Early pioneering work on measurements of the magnitude of the force required to create indentations with defined depth on their surfaces and to separate interacting pairs of avidin-biotin, antigen-antibody, and complementary DNA pairs formed the basis of this field. The method has subsequently been applied to map the presence of cell surface receptors and polysaccharides on live cell membranes by force measurement, with promising results. Attempts to extract phospholipids and proteins from lipid bilayers and live cell surfaces have been reported, providing a new tool for the manipulation of cellular activities and biochemical analysis at the single-cell level. An increasing awareness of the effect of the pulling speed (nm/s or microm/s), or more accurately, the force loading rate (pN/s or nN/s) on the magnitude of the rupture force, has led researchers to construct energy diagrams of rupture events based on the parameters available from such studies. Information on such nature of the interplay of force and loading rate is vital for nanomanipulation of living cells and cell membranes. Some relevant work for membrane manipulation using other methods is also reviewed in relation to AFM-based methodology.     

Kinetics of degradation of dipalmitoylphosphatidylcholine (DPPC) bilayers as a result of vipoxin phospholipase A2 activity: an atomic force microscopy (AFM) approach

In this paper we used AFM as an analytical tool to visualize the degradation of a phospholipid bilayer undergoing hydrolysis of the vipoxin's PLA(2). We obtained time series images during the degradation process of supported 1, 2-dipalmitoylphosphatidylcholine (DPPC) bilayers and evaluated the occurrence and the growth rate of the bilayer defects. The special resolution of the AFM images allowed us to measure the area and the perimeter length of these defects and to draw conclusions about the kinetics of the enzyme reaction. Moreover, we also report for some unique characteristics discovered during the vipoxin's PLA(2) action. Experimentally for the first time, we observed the appearance and the growth of three-dimensional (3D), crystal-like structures within the formed defects of the degraded bilayer. In an effort to explain their nature, we applied bearing image analysis to estimate the volume of these crystals and we found that their growth rate follows a similar kinetic pattern as the degradation rate of the supported bilayer.     

Changes in the hyperelastic properties of endothelial cells induced by tumor necrosis factor-alpha

Mechanical properties of living cells can be determined using atomic force microscopy (AFM). In this study, a novel analysis was developed to determine the mechanical properties of adherent monolayers of pulmonary microvascular endothelial cells (ECs) using AFM and finite element modeling, which considers both the finite thickness of ECs and their nonlinear elastic properties, as well as the large strain induced by AFM. Comparison of this model with the more traditional Hertzian model, which assumes linear elastic behavior, small strains, and infinite cell thickness, suggests that the new analysis can predict the mechanical response of ECs during AFM indentation better than Hertz's model, especially when using force-displacement data obtained from large indentations (>100 nm). The shear moduli and distensibility of ECs were greater when using small indentations (<100 nm) compared to large indentations (>100 nm). Tumor necrosis factor-α induced changes in the mechanical properties of ECs, which included a decrease in the average shear moduli that occurred in all regions of the ECs and an increase in distensibility in the central regions when measured using small indentations. These changes can be modeled as changes in a chain network structure within the ECs.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Cell Visco-Elasticity Measured with AFM and Optical Trapping at Sub-Micrometer Deformations

The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.     

Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.     

Transient phase of adenosine triphosphate hydrolysis by myosin, heavy meromyosin, and subfragment 1.

The transient phase of adenosine triphosphate (ATP) hydrolysis (early burst) was investigated for myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) over a range of temperatures and pH's. The burst size at pH 8,20 degrees C, is 0.8-0.85, based on steady-state and transient measurements. The equilibrium constant for the enzyme-substrate to enzyme-product transition is 0.85 +/- 0.05. It is concluded that both myosin heads undergo the rapid hydrolysis step and that there are no significant differences for S-1 vs. HMM or myosin. The transient data are fitted reasonably well by a single rate process, but available evidence is consistent with some heterogeneity and a range of rate constants differing by a factor of two. At pH 6.9 and 3 degrees C, the burst size is 0.5 and the hydrolysis is slower than the configuration change measured by fluorescence. The results are consistent with the kinetic scheme (see article). The lower burst at low temperature and pH can be partly explained by a reduction in the equilibrium constant, K3, and ATP can be synthesized on the enzyme by a pH-temperature jump.     

The mechanism of the skeletal muscle myosin ATPase. II. Relationship between the fluorescence enhancement induced by ATP and the initial Pi burst.

A major question about the mechanism of the myosin ATPase is how much of the fluorescence change which accompanies the binding of ATP to myosin is due to the conformational change induced by ATP and how much is due to the subsequent hydrolysis of ATP in the initial Pi burst. Several laboratories have suggested that the maximal rate of the fluorescence change represents the rate of the irreversible conformational change induced by ATP. In the present study, the rate of irreversible ATP binding, the rate of the initial Pi burst, and the rate of the fluorescence enhancement were compared under varied conditions. The results show that: 1) the fluorescence enhancement is mainly due to the hydrolysis of ATP in the initial Pi burst rather than to the conformational change induced by the binding of ATP; 2) the rate of the initial Pi burst is considerably slower than the rate of irreversible ATP binding at high ATP concentration; 3) the rate of the initial Pi burst is almost the same as the rate of the fluorescence enhancement. Therefore, the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.     

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.     

Fluid Secretion in Rhodnius Upper Malpighian Tubules (UMT): Water Osmotic Permeabilities and Morphometric Studies

We have measured the osmotic permeability of the basolateral cell membrane (Poscb) and compared it with the transepithelial permeability (Poste) to calculate the paracellular (Posp) permeability of the upper malpighian tubules (UMT) of the 5th instar of Rhodnius prolixus under several experimental conditions, namely, at rest and after stimulation to secrete with 5-HT, each under control conditions (no treatment), after treatment with pCMBS, and after addition of pCMBS and DTT. Secretion rate is negligible at rest. During stimulation mean secretion rate is 43.5 nl/cm2 sec. Secretion is severely curtailed by pCMBS and fully restored by DTT. Poscb = 9.4 (resting, control); 5.8 (control + pCMBS); 10.7 (control + pCMBS + DTT); 20.6 (stimulated, control); 14.7 (stimulated + pCMBS); 49.1 (stimulated + pCMBS + DTT) (x10?4 cm3/cm2 sec Osm). Calculated Posp are higher than the transcellular permeability, Posc, at rest and after stimulation. Electron micrograph morphometry of UMT sections show that cells significantly decrease their volume after stimulation. Lateral intercellular space (LIS) and basolateral extracellular labyrinth (BEL) are barely discernible at rest. LIS and BEL are widely dilated in stimulated UMT. Thus, ions have restricted access to the deep and narrow basolateral cell membrane indentations at rest, but they have ready access to cell membrane indentations after stimulation, because of the opening of LIS and BEL. These findings are discussed in relation to isosmotic secretion. The rate-limiting step for paracellular movement is located at the smooth septate junctions.     

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Molecular mechanism of GTP hydrolysis by bovine transducin: pre-steady-state kinetic analyses.

During the visual transduction process in rod photoreceptor cells, transducin (T) mediates the flow of information from photoexcited rhodopsin (R*) to the cGMP phosphodiesterase (PDE) via a cycle of GTP binding and hydrolysis. The pre-steady-state kinetics of GTP hydrolysis by T was studied by rapid quenching and filtration techniques in a reconstituted system containing purified R* and T. Kinetic analyses have shown that the turnover of T-bound GTP can be dissected into four partial reactions: (1) the R*-catalyzed GTP binding via a GDP/GTP exchange reaction, (2) the on-site hydrolysis of bound GTP, which leads to the formation of a T-GDP.Pi complex, (3) the release of the tightly bound inorganic phosphate (Pi) from T-GDP.Pi, and (4) the recycling of T-GDP. The R*-catalyzed GTP binding was estimated to occur in less than 1 s. In rapid acid quenching experiments, the rate of Pi formation due to GTP hydrolysis exhibited biphasic characteristics. An initial burst of Pi formation occurred between 1 and 4 s, which was followed by a slow steady-state rate. Increasing T concentration yielded a proportional increase in the burst and steady-state rate. The addition of Gpp(NH)p decreased both parameters. D2O decreased the rise of the initial burst with a kinetic isotope effect of approximately 1.7 but has no effect on the steady-state rate of Pi formation. These results indicate that the burst represents the fast hydrolysis of GTP at the binding site of T, which results in the accumulation of T-GDP.Pi complexes. The steady-state rate represents the slow release of Pi. This finding was further supported by rapid filtration experiments that monitored the formation of free Pi in solution. An initial lag time in the formation of free Pi was observed before a steady-state rate was established, indicating that the initially formed Pi was tightly bound to T. Finally, the release of GDP from T-GDP.Pi was not detected. This suggests that another cycle of GTP exchange catalyzed by R* should occur before the release of bound GDP. The rate of Pi release from T-GDP.Pi was measured under single-turnover conditions and had a half life of approximately 20 s, which was identical with the rate of deactivation of the PDE due to GTP hydrolysis by T.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.     

Enzymatic degradation of poly(L-lactide) film by proteinase K: quartz crystal microbalance and atomic force microscopy study

Enzymatic degradation of the poly(L-lactide) (PLLA) amorphous film by proteinase K has been investigated by combination of the complementary techniques of quartz crystal microbalance and atomic force microscopy (AFM). The erosion rate increased with increasing enzyme concentrations and attained to be constant under the condition of [proteinase K] > 100 microg/mL. The amount of the enzyme molecules adsorbed to the film was quantitatively evaluated at various concentrations by AFM, and it revealed that the erosion rate is determined by the amount of adsorbed enzyme. Adsorption of proteinase K was irreversible despite lack of the binding domain, so that the enzyme molecules on the film surface could be observed directly by AFM. Transformation of the enzyme molecule caused by packing in high density on the surface was observed at higher enzyme concentrations. The "footprint" of the individual proteinase K molecule on the PLLA film after enzymatic degradation suggests that the enzyme moves on the surface to hydrolyze the film around it.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.