Phosphate Release Research Articles

Impact of Germination on the Protein Solubility and Antinutritive Compounds of Lupinus angustifolius and Vicia faba in the Production of Protein-Rich Legume-Based Beverages.

Lupines and faba beans are protein-rich legumes, which can be utilized as a plant-based substitute for animal proteins in human nutrition in general and in the beverage industry in particular. However, their application is hampered by low protein solubility in the acidic pH range and by antinutrients such as flatulence-causing "raffinose family oligosaccharides" (RFOs). Germination is known in the brewing industry for increasing enzymatic activities and mobilizing storage compounds. Therefore, germinations of lupines and faba beans were performed at different temperatures, and their impact on protein solubility, the concentration of free amino acids, and degradation of RFOs, alkaloids, and phytic acid was evaluated. In general, changes were comparable for both legumes but less pronounced in faba beans. Germination depleted the RFOs entirely in both legumes. The protein size distribution was found to be shifted toward smaller fractions, the concentrations of free amino acids multiplied, and protein solubility increased. No substantial reductions in the binding capacity of phytic acid toward iron ions were observed, but a release of free phosphate in lupines was detected. The results prove that germination is an applicable process for refining lupines and faba beans for use not only in refreshing beverages or milk alternatives but also in other food applications.

A mutation in switch I alters the load-dependent kinetics of myosin Va

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (Pi) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated Pi on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of Pi-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM Pi, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of Pi from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the Pi-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of Pi-rebinding.

Quantification of Nutrient Fluxes from Sediments of Lake Hulun, China: Implications for Plateau Lake Management

Dramatic changes in lake water conditions may induce sensitive feedback in sediments, such as the release of phosphate and ammonia. There is a lack of the understanding of sediment nutrient release fluxes and their responses to various environmental factors in plateau lakes. In this study, nutrient contents in the water and sediment, sediment nutrient release rates, and their responses to various influential factors in a steppe shallow plateau lake, Lake Hulun, China, were investigated. Sediment is a large store of nutrients, with bioavailable phosphorus of 555 mg kg−1 in surface lake sediments. If water became anoxic, sediments will release appreciable amounts of phosphate (2.1 mg P m−2 d−1) and ammonia (40.0 mg N m−2 d−1), three times greater than those in oxic conditions. Raised temperatures greatly increased sediment phosphate release rates, while the highest temperature (23 °C) slightly decreased ammonia release rates due to strong nitrification. Assuming a whole year of internal loading, ammonia and dissolved reactive phosphorus concentrations in water column would increase by an estimated 0.22 mg N L−1 and 0.01 mg P L−1, respectively, which is equal to 2 and 0.5 times of their ambient concentrations. A pH increase from 8.4 to 9.4 significantly decreased internal nitrogen and phosphorus release rates by around three times. Overall, dissolved oxygen (DO) and pH had a greater impact on sediment nutrient release from lake sediments compared to temperature, and further increased the eutrophic status in the overlying water when these three parameters varied within the normal seasonal range (temperature: 1.9–23.1 °C, DO: oxic and anoxic; pH: 7.5–9.4). With the periodic low DO and frequency of drought in the future, lake management strategies that mitigate water eutrophication, such as water diversion, may need to be considered.

Impacts of calcium peroxide on phosphorus and tungsten releases from sediments

In this study, CaO2 was used as a capping material to control the release of Phosphate (P) and tungsten (W) from the sediment due to its oxygen-releasing and oxidative properties. The results revealed significant decreases in SRP and soluble W concentrations after the addition of CaO2. The mechanisms of P and W adsorption by CaO2 were mainly chemisorption and ligand exchange mechanisms. In addition, the results showed significant increases in HCl–P and amorphous and poorly crystalline(oxyhydr)oxides bound W after the addition of CaO2. The highest reduction rates of sediment SRP and soluble W release were 37 and 43%, respectively. Furthermore, CaO2 can promote the redox of iron (Fe) and manganese (Mn). On the other hand, a significant positive correlation was observed between SRP/soluble W and soluble Fe (II) and between SRP/soluble W and soluble Mn, indicating that the effects of CaO2 on Fe and Mn redox play a crucial role in controlling P and W releases from sediments. However, the redox of Fe plays a key role in controlling sediment P and W release. Therefore, CaO2 addition can simultaneously inhibit sediment internal P and W release.

Boron nitride nanofiber/Zn-doped hydroxyapatite/polycaprolactone scaffolds for bone tissue engineering applications.

In this study, Zn doped hydroxyapatite (Zn HA)/boron nitride nanofiber (BNNF)/poly-ε-caprolactone (PCL) composite aligned fibrous scaffolds are produced with rotary jet spinning (RJS) for bone tissue engineering applications. It is hypothesized that addition of Zn HA and BNNF will contribute to cell viability as well as mechanical and osteogenic properties of the PCL scaffolds. Zn HA was synthesized by mixing Ca and P sources followed by sonication and aging whereas BNNF was obtained by the reaction of melamine with boric acid followed by freeze-drying for annealing of fibers. It is found that incorporation of both Zn HA and BNNF in PCL fibers resulted in higher calcium phosphate (CaP) precipitation on the scaffolds. Also, in vitro cell culture studies showed that presence of both Zn HA and BNNF also had synergistic effect for enhanced proliferation and osteogenic activity of Saos-2 cells. Mechanical properties of PCL-Zn HA-BNNF were found similar to that of non-load bearing bones. Furthermore, the presence of Zn HA and BNNF had synergistic effects to cell attachment, proliferation and spreading without causing cytotoxic effect on cells. The highest ALP activity was obtained in the PCL-Zn HA- BNNF group at days 7 and 14 due to release of zinc, calcium, phosphate and boron. Considering its mechanical and bioactivity properties, PCL-Zn HA-BNNF composite scaffolds hold promise as non-load bearing bone substitutes.

A selective and atom-economic rearrangement of uridine by cascade biocatalysis for production of pseudouridine

As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ−5’-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of β-d-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via d-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5’-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼105 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.

DNA hydrogels for bone regeneration

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions invitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth invitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels invitro, as characterized by fluorescence microscopy. Invivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

Phosphatase activity of Phosphate Solubilizing Fungi Isolation from Rhizosphere Soil of Medicinal Plants of Shivamogga District

Phosphatases are a group of enzymes which significantly accelerate the release of inorganic phosphate from organically bound phosphate and return to the soil by phosphate solubilizing fungi. Hence for the present study, we focused on the activity of phosphatase. Total 46 fungi were isolated from 55 rhizosphere soils of medicinal plants at Malnad regions of Shivamogga district. Among them, 10 isolates were selected by screening them using their solubilization index ranging from 3.02 to 3.96 and they were further evaluated. The selected 10 isolates showed good results in phosphate solubilization parameters. Decreased pH was observed in culture broth (4.3 to 3.0) from initial pH of 6.89. Amount of acid present in the culture broth (30.01 - 38.08g/L) and concentration of phosphate (70 - 20μg/ml) were recorded. The phosphatases activity was determined on 7th, 14th, 21st and 28th days after incubation at room temperature. Among them, PSF 44 (Aspergillus awamori) showed maximum alkaline phosphatase activity of 0.81, 1.53, 2.15 and 2.38μg PNP liberated/ml/min and acid phosphatases activity of 0.71, 1.34, 2.30 and 2.34μg PNP liberated/ml/min on 7th, 14th, 21st and 28th day respectively after incubation. Hence due to the maximum phosphatase activity and phosphate solubilization capacity of Aspergillus awamori, they can be recommended as phospho-fungal bioinoculum for agricultural field.

Impact of borosilicate bioactive glass scaffold processing and reactivity on in-vitro dissolution properties

In this study, bulk borosilicate glasses and 3D scaffolds (processed by the burn-off technique and by robocasting) were synthesized to investigate the impact of the manufacturing method, glass composition and preincubation time on in vitro dissolution and cell response. The studied compositions are based on commercial bioactive glass S53P4 (BonAlive) where 12.5% SiO2 has been replaced by B2O (labelled B12.5), and part of the CaO is replaced with MgO and SrO (labelled B12.5-Mg-Sr). First, the impact of the processing and glass composition, on the dissolution rate, was assessed. As expected, scaffolds were found to exhibit faster dissolution, due to the increased surface area, when compared to the bulk glass. Furthermore, the 3D printed scaffolds were found to dissolve faster than the burn-off scaffolds. Moreover, scaffolds made from B12.5-Mg-Sr glass composition exhibited slower ion release and precipitation of calcium phosphate (CaP) layer, when compared to B12.5, due to the stabilizing effect of Mg and Sr. Finally, dynamic condition produces lower ion releases that static condition and could be more optimal for in vitro cell growth. Secondly, in culture with murine MC3T3-E1 cells, it was shown that 3 days preincubation would be optimal to decrease the burst of ions that is known to lead to cell death. However, it was found that MC3T3-E1 survived and proliferated only in presence of B12.5-Mg-Sr scaffolds. Finally, it was shown that despite scaffolds having different porosities, they had no significant difference on human adipose-derived stem cells (hADSCs) survival. This manuscript brings new information on 1) the impact of material design (porosity) and composition on dissolution kinetic sand reactivity, 2) the impact of static vs dynamic testing on in-vitro dissolution and 3) the impact of materials’ pre-incubation on cell behavior.

New insights into the mechanism of phosphate release during particulate organic matter photodegradation based on optical and molecular signatures

Phosphate release from particulate organic matter (POM) dominates phosphorus (P) cycling in aquatic ecosystems. However, the mechanisms underlying P release from POM remain poorly understood because of complex fractionation and analytical challenges. In this study, the release of dissolved inorganic phosphate (DIP) during POM photodegradation was assessed using excitation–emission matrix (EEM) fluorescence spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). POM in suspension was significantly photodegraded under light irradiation, concomitantly with the production and release of DIP in the aqueous solution. Chemical sequential extraction revealed that organic phosphorus (OP) in POM participated in photochemical reactions. Moreover, FT-ICR MS analysis revealed that the average molecular weight of P-containing formulas decreased from 374.2 to 340.1 Da. Formulas containing P with a lower oxidation degree and unsaturation were preferentially photodegraded, generating oxygen-enriched and saturated formula compounds, such as protein- and carbohydrate-like P-containing formulas, benefiting further utilization of P by organisms. Reactive oxygen species played an important role in the photodegradation of POM, and excited triplet state chromophoric dissolved organic matter (3CDOM*) was mainly responsible for POM photodegradation. These results provide new insights into the P biogeochemical cycle and POM photodegradation in aquatic ecosystems.

Enzyme‐assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate

Background The analysis of myo-inositol phosphates (InsPx) released by phytases during phytic acid degradation is challenging and time-consuming, particularly in terms of sample preparation, isomer separation, and detection. However, a fast and robust analysis method is crucial when screening for phytases during protein engineering approaches, which result in a large number of samples, to ensure reliable identification of promising novel enzymes or target variants with improved characteristics, for example, pH range, thermal stability, and phosphate release kinetics. Results The simultaneous analysis of several InsPx (InsP1-InsP4 and InsP5 + 6) as well as free phosphate was established on cellulose HPTLC plates using a buffered mobile phase. Inositol phosphates were subsequently stained using a novel enzyme-assisted staining procedure. Immobilized InsPx were hydrolyzed by a phytase solution of Quantum® Blueliquid 5G followed by a molybdate reagent derivatization. Resulting blue zones were captured by DAD scan. The method shows good repeatability (intra-day and intra-lab) with maximum deviations of the Rf value of 0.01. The HPTLC method was applied to three commercially available phytases at two pH levels relevant to the gastrointestinal tract of poultry (pH 5.5 and pH 3.6) to observe their phytate degradation pattern and thus visualize their InsPx fingerprint. Conclusion This HPTLC method presents a semi-high-throughput analysis for the simultaneous analysis of phytic acid and the resulting lower inositol phosphates after its enzymatic hydrolysis and is also an effective tool to visualize the InsPx fingerprints and possible accumulations of inositol phosphates.

Eco-Friendly Starch Composite Supramolecular Alginate-Ca2+ Hydrogel as Controlled-Release P Fertilizer with Low Responsiveness to Multiple Environmental Stimuli.

Environmentally friendly fertilizers (EFFs) have been developed to improve fertilizer efficiency and minimize adverse environmental impacts, but their release behavior under various environmental conditions has been less explored. Using phosphorus (P) in the form of phosphate as a model nutrient, we present a simple method for preparing EFFs based on incorporating the nutrient into polysaccharide supramolecular hydrogels using Cassava starch in the Ca2+-induced cross-link gelation of alginate. The optimal conditions for creating these starch-regulated phosphate hydrogel beads (s-PHBs) were determined, and their release characteristics were initially evaluated in deionized water and then under various environmental stimuli, including pH, temperature, ionic strength, and water hardness. We found that incorporating a starch composite in s-PHBs at pH = 5 resulted in a rough but rigid surface and improved their physical and thermal stability, compared with phosphate hydrogel beads without starch (PHBs), due to the dense hydrogen bonding-supramolecular networks. Additionally, the s-PHBs showed controlled phosphate-release kinetics, following a parabolic diffusion with reduced initial burst effects. Importantly, the developed s-PHBs exhibited a promising low responsiveness to environmental stimuli for phosphate release even under extreme conditions and when tested in rice field water samples, suggesting their potential as a universally effective option for large-scale agricultural activities and potential value for commercial production.

Co-pyrolysis of biomass with magnesium and phosphorus: effect of magnesium content on phosphate release from biochar-based fertilizers

Co-pyrolysis of biomass with magnesium and phosphorus: effect of magnesium content on phosphate release from biochar-based fertilizers

Highly efficient removal of phosphonates by ferrate-induced oxidation coupled with in situ coagulation

Phosphonates, as a kind of important organic phosphorus in wastewater, should be removed in terms of their environmental risks. Unfortunately, traditional biological treatments fail to remove phosphonates effectively due to their biological inertness. The reported advanced oxidation processes (AOPs) usually require pH adjustment or coupling with other technologies to achieve high removal efficiency. Thus, a simple and efficient method for phosphonate removal is urgently needed. Herein, ferrate was found to remove phosphonates effectively in one-step under near-neutral circumstances by coupling oxidation and in-situ coagulation. Nitrilotrimethyl-phosphonic acid (NTMP), a typical phosphonate, could be efficiently oxidized by ferrate to release phosphate. The fraction of phosphate release increased with increasing ferrate dosage and reached 43.1% when 0.15 mM ferrate was added. Fe(VI) was responsible for NTMP oxidation, while Fe(V), Fe(IV) and ⋅OH played a minor role. Ferrate-induced phosphate release facilitated total phosphorus (TP) removal, because the phosphate is more easily removed via ferrate-resultant Fe(III) coagulation than the phosphonates. The coagulation removal of TP could reach up to 90% within 10 min. Furthermore, ferrate exerted high removal efficiencies for other commonly used phosphonates with approximately or up to 90% TP removal. This work provides a one-step efficient method to treat phosphonate-containing wastewaters.

Waste hemp-stalk derived nutrient encapsulated aerogels for slow release of fertilizers: A step towards sustainable agriculture

With a renewed focus towards circular economy, a novel eco-friendly aerogel was prepared from waste hemp stalk derived cellulose for sustained release of fertilizers. The approach encompasses replenishment the bio-waste back to the soil with addition of fortifying nutrients. Cellulose was converted to carboxymethyl cellulose (HCMC), which was further cross-linked using a green moiety, citric acid (CA) to a yield a three-dimensional aerogel (HCMC-CA-aero). Chemical, physical and thermal properties of the aerogel were evaluated using FTIR, XRD, TEM, EDS, FESEM and TGA. The HCMC-CA-aero was assessed for its swelling behavior, pH response and fertilizer release mechanism, both in water and soil. The aerogel absorbed 80 g/g water after 27 h at neutral pH exhibiting super-absorbent behavior owing to its highly porous structure, large free volume available amid polymeric chains, occurrence of large number of hydrophilic groups and high flexibility. It also exhibited a sustained release of encapsulated nutrients, urea and ammonium dihydrogen phosphate (ADP). The percentage release of nutrients in water after 40 h was 48.4 % and 44.46 % for HCMC-CA-aero@urea and HCMC-CA-aero@ADP, respectively, while in soil it was 62.28 % and 56.62 % after 120 h, unveiling its excellent potential for soil conditioning. The release kinetics for both the fertilizers followed Higuchi model and Fickian diffusion, demonstrating a controlled release of fertilizers governed by dissolution and diffusion. The results indicated that this is an effective approach for agriculture waste management and has great potential in sustained agriculture applications.

A rare case of tumour lysis syndrome in multiple myeloma

Tumour lysis syndrome (TLS) is a serious life-threatening complication of cancer chemotherapy which is a constellation of metabolic disturbances that typically occurs during therapy of bulky, rapidly proliferative tumours. TLS is an oncological emergency that causes a significant release of phosphate, potassium, uric acid, and nucleic acids into the blood stream. Incidence of TLS in multiple myeloma is rare. Here we present a case of multiple myeloma who later developed tumour lysis syndrome in its due course which was rare. A 67-year-old male was diagnosed with Multiple myeloma on the basis of M spike, osteolytic lesions and bone marrow analysis. First cycle of chemotherapy with lenalidomide, bortezomib and denosumab was initiated. During the course of follow up he presented to our casualty with complaints of chills and rigor. Based on the biochemical abnormalities a diagnosis of tumour lysis syndrome was made. He received I.V fluids, anti-hyperkalemic measures, allopurinol. He had neutropenic sepsis and went into septic shock and died after multiple resuscitative efforts.

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

Optimization of the anaerobic fermentation process for phosphate release using food waste

Phosphorus (P) problem worries the whole world due to the increasing demand for finite and non-renewable natural phosphate resources and the inadequacy of sustainable phosphate production technologies. In this study, bio-acidification processes using waste sludge and food waste for simultaneous sustainable phosphate release and biogas production were investigated. Response surface methodology (RSM) was used for bio-acidification optimization. High performance was achieved with the addition of 10% FW and a temperature of 45 °C, which provided 5.30 pH and 371 mg/L P release for 10 days. A total of 196 mL of cumulative biogas was produced. Using food waste potentially reduces operating costs, eliminating the need for external chemical additions for pH control. Also, this approach offers benefits such as waste management, recovery of valuable resources, cost reduction, and environmental friendly.

A point mutation in switch 1 alters the load dependence of phosphate rebinding to actomyosin.

Myosin transduces chemical energy from ATP into mechanical work to generate force and/or motion. Key to this process is the coupling of the powerstroke and the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved remain unclear. We determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10 pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30 mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7 nm for the WT myosin. Repeating these experiments using a construct with a mutation in the Switch I region of the active site (S217A) increased myosin's sensitivity to load five-fold (d = 3.5 nm). Thus, these findings provide a quantitative measure of the force-dependence of Pi-rebinding to myosin and suggest that this effect involves the switch I element of the nucleotide-binding pocket.

Solution kinetics analysis of vesicle trafficking of zymogen granule.

Zymogen granule (ZG) is a micrometer size enzymatic vesicle in the exocrine pancreas. It regulates secretion of digestive enzymes. It is also associated with pancreatic disease and disorder. It has several myosin classes molecular motors such as 1c, 5c, 6 and 7b. These myosin motors are responsible for transportation of ZG. The hydrolysis of adenosine triphosphate (ATP) initiates the directional motion of these myosin motors. To understand what the key part of the kinetics step for the ZG's trafficking, we measured each step of ATPase of purified ZGs from the rat pancreas. In the present study, we employed the stop flow techniques in order to investigate step(s) of ATPase cycle, e.g. ATP binding, ADP dissociation, phosphate release, and actin binding rate. The data were observed as ensemble work of myosins. The results provide the kinetics of these molecular motors complex present in the ZG. For ATP binding, mant-dATP was used, and result was fit with a single exponential with about 200 (/s) rate. For ADP dissociation, mant-dADP was used with double mixing methods. The fast and slow rates of ADP dissociation are 120 /s and 28 /s, respectively. For actin binding rate, Pyrene-actin was used. The fitting curves were found to be double exponential rates. The fast and slow rates are 64 /s and 12 /s, respectively. For the phosphate release, Phosphate binding protein was used and was observed to be double exponential, and the fast and slow rates are 250 /s and 8/s, respectively. Comparing only Myosin 5c, slow rate part of ADP dissociation and phosphate dissociation shows a little more significance, while ATP binding rate is not different.