Extraction Of Myosin Research Articles

L-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

L‐Arginine and l‐lysine can weaken the intermolecular interactions of main myofibrillar proteins: the roles in improving the tenderness of pork Longissimus lumborum muscle

SummaryThis work aims at investigating the mechanisms underlying the effects of l‐arginine and l‐lysine on the tenderness of pork Longissimus lumborum muscle. The results indicated that on day 7, L‐arginine and l‐lysine were able to decrease the shear force from 71.47 to 27.59 or 32.53 N, but raise the myofibrillar fragmentation index from 70.93 to 88.2 or 92.4, compared to the control. Additionally, l‐arginine inhibited the degradation of desmin, troponin‐T and titin; while l‐lysine inhibited the degradation of desmin, troponin‐T, titin and nebulin. l‐Arginine, especially l‐lysine alleviated the Z‐disc damage, while accelerated the M‐line damage. l‐Arginine and l‐lysine promoted the extraction of myosin, titin and some unidentified proteins, decreased ionic bonds but increased hydrophobic interactions. The results suggest that the weakening of the intermolecular interactions between main myofibrillar proteins by l‐arginine or l‐lysine may play crucial roles in improving meat tenderness. The finding may broaden the way to tenderise meat.

The Evolution of The Cardiac Myosinome of Gnathostomes

The term myosinome has been recently coined to define the set and distribution of myosins in a particular tissue. Since different myosin isoforms have different contractile properties, the myosinome of cardiac fibers determines their type of contraction. Classically, it has been assumed that the cardiac myosinome of vertebrates is mainly formed by only two myosin heavy chain (MYH) isoforms, which are heterogeneously distributed in the heart: whereas MYH6 is more abundant in the inflow, MYH7 predominates in the outflow myocardium. However, this concept has been recently questioned after the identification of significant amounts of additional isomyosins in hearts of other vertebrates, i.e. MYH15 in the amphibian outflow myocardium; MYH2 and MYH7B in the inflow and outflow myocardia of chondrichthyans, respectively. To study the diversity and evolution of the cardiac myosinome in vertebrates, we have analyzed the inflow and outflow myocardia of representative specimens of polypteriforms, lepisosteiforms, acipenseriforms and dipnoi, by means of a specific myosin extraction method followed by quantitative proteomic analysis. We have identified five additional MYH isoforms (MYH1, MYH3, MYH4, MYH8 and Fast-skeletal MYH), each abundantly expressed in the inflow or outflow myocardia of distinct vertebrate groups, demonstrating that the cardiac myosinome in vertebrates is more diverse than initially thought. In general, the diversity of MYH isoforms has increased in most osteichthyes and decreased in teleosts and tetrapods. Therefore, not all vertebrates share the MYH6/MYH7 pattern observed in teleosts, birds and mammals, which can be considered a specialized vertebrate myosinome. In addition, the abundance and distribution of these MYH isoforms in the morpho-functionally diverse hearts studied allow raising several hypotheses on the evolution of the vertebrate cardiac myosinome: While MYH4 seems to be an autapomorphy of non-tetrapod sarcopterygians, MYH7B can be considered an ancient myosin, only present in hearts with a primitive anatomical design. The primitive type of cardiac contraction in gnathostomes with a linear heart anatomy and a long conus arteriosus might require the expression of MYH7B together with MYH1 and Fast-skeletal MYH. Cardiac MYH2 expression may have appeared in association with the development of a specialized cardiac conduction system, whereas that of MYH3 and MYH8 might be associated with adaptation to environments with low oxygen tension. Further studies are needed to confirm these hypotheses on the evolution of the cardiac myosinome, which constitutes a new research field on cardiovascular evolution.

What makes skeletal muscle striated? Discoveries in the endosarcomeric and exosarcomeric cytoskeleton.

One of the most iconic images in biology is the cross-striated appearance of a skeletal muscle fiber. The repeating band pattern shows that all of the sarcomeres are the same length. All of the A bands are the same length and are located in the middle of the sarcomeres. Furthermore, all of the myofibrils are transversely aligned across the muscle fiber. It has been known for 300 yr that skeletal muscle is striated, but only in the last 40 yr has a molecular understanding of the striations emerged. In the 1950s it was discovered that the extraction of myosin from myofibrils abolished the A bands, and the myofibrils were no longer striated. With the further extraction of actin, only the Z disks remained. Strangely, the sarcomere length did not change, and these "ghost" myofibrils still exhibited elastic behavior. The breakthrough came in the 1970s with the discovery of the gigantic protein titin. Titin, an elastic protein ~1 µm in length, runs from the Z disk to the middle of the A band and ensures that each sarcomere is the same length. Titin anchors the A band in the middle of the sarcomere and may determine thick-filament length and thus A-band length. In the 1970s it was proposed that the intermediate filament desmin, which surrounds the Z disks, connects adjacent myofibrils, resulting in the striated appearance of a skeletal muscle fiber.

An historical perspective of the discovery of titin filaments.

This review takes readers back to 1949, when two Australian scientists, Draper and Hodge, reported the first high-resolution electron microscopy images of striated muscle. In 1953, Jean Hanson and Hugh Huxley published phase-contrast microscopy and electron microscopy images that established the filamentous nature of the sarcomere, namely the myosin-containing thick filaments and actin-containing thin filaments. They discussed a putative third filament system, possibly a thinner actin-containing S filament, that appeared to connect one Z disc to the next. The next year, two back-to-back papers appeared in Nature, the first by Andrew Huxley and Rolf. Niedergerke, the second by Hugh Huxley with Jean Hanson. Independently, they proposed the sliding of actin filaments and myosin filaments. These two filaments quickly became firmly established in the literature and, even today, they remain the basis for the sliding filament hypothesis. The putative third filament concept was dropped, mainly through the lack of evidence but also because it was difficult to accommodate in the hypothesis where two sets of filaments maintained their lengths constant while sliding produced sarcomere shortening. The view that actin and myosin comprise more than 80% of the myofibril proteins also made it difficult to accommodate a major new protein. In the following years, using selective extraction of myosin and actin, dos Remedios (PhD thesis, University of Sydney, 1965) revealed a residual filament system in the sarcomere, and, once again, a third filament system re-entered the literature. Filaments were reported crossing the gap between the ends of thick and thin filaments in highly stretched muscle fibres. These and other early studies necessarily focussed on light and electron microscopy, and set the scene for investigations into the chemical nature and biophysical functions of the third filament system for striated muscles. Further progress had to wait for the improvement and/or development of a number of techniques. For example, in 1970, Laemmli (Nature 227:680-685, 1970) published an often cited method for improving SDS polyacrylamide gel electrophoresis. The Lowry et al. (J Biol Chem 193:265-275, 1951) protein assay method developed in 1950 was both unstable and insensitive in comparison, but we had to wait until 1976 for the development of the Bradford method (1976). Atomic force microscopy was not known before 1986, but it eventually enabled the direct measurement of single molecules of titin. This extraordinarily large (>106Da) elastic protein became known as connectin (Maruyama in J Biochem 80:405-407, 1976) and was subsequently named titin (Wang et al. in Proc Natl Acad Sci U S A 76:3698-3702, 1979). Prior to the discovery of titin/connectin, biophysicists found it difficult to understand howa single polypeptide chain could could stretch from the Z disc to the M line, a distance of more than 1μm. It was quite literally the 'elephant in the room'. In this review, we follow the trail of microscopy-based reports that led to the emergence of what is now known and accepted as titin, an elastic third filamentous protein that is the focus of this Special Issue.

Different actions of salt and pyrophosphate on protein extraction from myofibrils reveal the mechanism controlling myosin dissociation.

Myosin is the major functional protein in muscle foods for water retention, protein binding/gelation and fat holding/emulsification. To maximize its functionality, myosin needs to be released from thick filaments. Understanding of the mechanism controlling myosin extraction will help improve quality traits of meat products. The data obtained show that actomyosin binding is the rate-limiting constraint for myosin release in rigor condition. Magnesium pyrophosphate (MgPPi) increased myosin extraction by weakening actomyosin interaction and maximized myosin extraction at 0.4 mol L(-1) NaCl, which was not attained at 1.0 mol L(-1) NaCl in the absence of PPi. Interaction between myosin rod domains is another critical constraint for myosin extraction, which is, rather than PPi, salt dependent. Further, our data suggest that MyBP-C (myosin binding protein C) and M-line might not be of significance in the process of NaCl-induced myosin extraction, though further study was needed. Our study provides new insight into the mechanism that controls myosin extraction from intact sarcomere, which could be applied to maximize myosin function and to improve meat quality in practice.

Enhancing Effect of IMP on Myosin and Actin Extraction from Porcine Meat

We examined the effects of nucleoside monophosphates on the dissociation of actomyosin into myosin and actin. GMP was effective only among GMP, CMP, dTMP, and UMP. Hence we concluded that purine-based nucleoside monophosphates such as GMP, AMP, and IMP are effective, incorporating this with our previous results (Okitani A et al., Biosci. Biotechnol. Biochem., 72, 2005-2011 (2008)). Then we examined whether IMP enhances the extraction of myosin and actin as well as pyrophosphate (KPP), using homogenates of pork with 9 volumes of 0.3, 0.4, and 0.5 M NaCl solutions containing 0-36 mM IMP or 0-9 mM KPP. Maximum extractability, about 70% for both proteins, was attained by means of NaCl solutions containing 36 mM IMP. These values were comparable to the maximum values, about 90% for myosin and 50% for actin, attained by means of solutions containing 9 mM KPP. Hence we concluded that IMP enhances the extraction of myosin and actin from porcine meat.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle.

A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ.With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4-30◦C) and substrate concentration. VF was 8.88 ± 0.26 μms⁻¹ at 30.6◦C and decreased to 1.60 ± 0.09 μms⁻¹ at 4.5◦C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40-50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5◦C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.

Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin

The kinetics of myosin dissociation from actin was investigated and also the impact of salt, MgPPi, and myosin heavy chain isoform on myosin subfragment 1 (S1) dissociation from actin using purified proteins and fluorescence spectroscopy. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentrations increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine masseter (slow) and cutaneous trunci (fast) muscle increased 38 and 78 fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin. With the addition of MgPPi to the mixture of pyrene actin and S1, the fluorescence increased about 85% within the dead time of the mixing approach.. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin. The results reveal that salt and MgPPi increase myosin extraction and functionality in meat by weakening the actomyosin interaction and that some of the difference in the functionality of red and white muscle may be related to actomyosin dissociation.

In Vitro Study of Mechanical and Kinetic Properties of Myosin II from Frog Skeletal Muscle

We provide for the first time the protocol for efficient extraction and conservation of myosin II from frog skeletal muscle, a methodological achievement that makes it possible to apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic characteristics in single muscle cells, where it works as an ensemble in each half-sarcomere. With the in vitro motility assay, we estimate the sliding velocity of actin on frog myosin II (VF) and its modulation by temperature (range 4-30 °C) and substrate concentration. VF is 8.88 ± 0.511 μm/s at 30.6 °C and decreases down to 1.6 ± 0.23 μm/s at 4.5 °C. The in vitro mechanical and kinetic parameters are integrated with the in situ mechanical and kinetic parameters of frog muscle myosin working in array in each half-sarcomere. By comparing VF with the shortening velocity determined in intact frog muscle fibres under different loads and their dependence on temperature (Piazzesi et al., J. Physol., 549:93, 2003), we find that VF is 40-50% less than the in situ unloaded shortening velocity (V0) at the same temperature and we determine the load that explains the reduced value of VF. With the integrated approach we can define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we clarify the relation between the rate of ADP release and the rate of detachment of myosin from actin and their temperature dependence. Supported by NIH (Grant no. 5R01AR49033) and MiUR Italy.

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.

Characterization of the Myosin-Based Source for Second-Harmonic Generation from Muscle Sarcomeres

Several biologically important protein structures give rise to strong second-harmonic generation (SHG) in their native context. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information based on the physical constraints of the optical effect. In this study we characterize, by biochemical and optical analysis, the critical structures underlying SHG from the complex muscle sarcomere. SHG emission arises from domains of the sarcomere containing thick filaments, even within nascent sarcomeres of differentiating myocytes. SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. Furthermore, the polarization dependence of sarcomeric SHG is not affected by either the proportion of myosin head domains or the orientation of myosin heads. By fitting SHG polarization anisotropy readings to theoretical response curves, we find an orientation for the elemental harmonophore that corresponds well to the pitch of the myosin rod α-helix along the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving SHG from striated muscle. This study should guide the interpretation of SHG contrast in images of cardiac and skeletal muscle tissue for a variety of biomedical applications.

Calcium‐induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.

Role of myofibrillar proteins in water-binding in brine-enhanced meats

Brine-incorporated meats have become major novelty food items in the present fresh meat retail market in the United States. Brines are used to “enhance” products’ moistness and juiciness; however, the physicochemical processes involved in water-binding and entrapment in the meat protein matrix have not been completely elucidated. This paper describes our recent studies on the mode of water-binding in chicken and pork muscle foods following phosphate, NaCl, and enzyme-hydrolyzed soy protein brine treatments. The results showed that the dynamics of brine penetration into muscle fibrils were specific to phosphate types, with pyrophosphate and tripolyphosphate having the highest efficacy. Both phosphate treatments produced a transverse expansion of the myofibrils with a simultaneous extraction of myosin from the ends of the A-band in the sarcomere. These structural/biochemical changes resulted in substantial swelling of muscle fibers, i.e., an enhanced water uptake and immobilization. Furthermore, depending on the type of proteases employed, soy protein hydrolysates or peptides interacted differently with myofibrillar proteins, producing an array of morphologies and rheologies of protein gels that played a major role in water-immobilization in salted pork products.

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as ∼10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 μg/ml alamethicin and the reaction is terminated by H 2SO 4, which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1–20 μM. NADH is quantitatively analyzed with a detection limit of 3–5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.

Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene, Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of the Act88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin. However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through F-actin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2-7) that actin residue Glu(93) is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary site.

Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.

Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.

Study of regulation of mitochondrial respiration in vivo: An analysis of influence of ADP diffusion and possible role of cytoskeleton

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the “ghost” (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent K m (low affinity) equal to 300–400 μM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN 3 did not decrease the apparent K m for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40–70 μM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent K m values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent K m for ADP and the second one with very low apparent K m for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent K m for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils.

Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.

Regulation of calmodulin binding to the ATP extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH) 2D 3

In earlier studies we observed that the active vitamin D metabolite 1,25-(OH) 2D 3 increased the calmodulin content of purified duodenal brush-border membrane vesicles where it bound principally to the 110 kDa protein myosin I. In this study we further evaluated the regulation of calmodulin binding to ATP releasable myosin I. Whole brush borders (BB) or purified brush-border membrane vesicles (BBMV) were prepared from duodena of vitamin D-deficient rachitic chicks treated 12–18 h before killing with either 625 pmol 1,25-(OH) 2D 3 or vehicle. The ATP extractable myosin I from BB resulted in an 1.6-fold increase of calmodulin binding to the 110 kDa band after treatment with 1,25-(OH) 2D 3. In contrast to BB, ATP extraction of myosin I from purified BBMV required alamethicin for ATP entry. As for BB extracts, calmodulin binding to the 110 kDa band in BBMV extracts was also increased about 2.4-fold by 1,25-(OH) 2D 3. It was concluded that both intact BB and purified BBMV showed the same type of increase in calmodulin binding to ATP releasable myosin I by 1,25-(OH) 2D 3. To see whether 1,25-(OH) 2D 3 increased the intrinsic affinity of calmodulin binding to myosin I, the ATP extractable myosin I from BB was purified from rachitic chicks treated with 1,25-(OH) 2D 3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmodulin binding to the purified myosin I was not different between preparations from 1,25-(OH) 2D 3- or vehicle-treated chicks. We conclude that 1,25-(OH) 2D 3 does not change the affinity of calmodulin binding to myosin I but increases the amount of myosin I in the membrane or alters its ATP releasability. It was further investigated whether phosphorylation is involved in these 1,25-(OH) 2D 3 dependent posttranslational changes of myosin I. Phosphorylation of brush-border membrane proteins in vivo was performed by incubation of [ 32P]P i in the lumen of a ligated duodenal loop in situ for 15 min. Brush-border membrane proteins were phosphorylated in vitro by incubating BB or BBMV with [γ- 32P]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulted in phosphorylation of several proteins including 110 kDa proteins. However, there was no specific effect of 1,25-(OH) 2D 3 on phosphorylation of 110 kDa proteins. We conclude that the effects of 1,25-(OH) 2D 3 on protein phosphorylation are minimal and not likely to explain 1,25-(OH) 2D 3 stimulated calmodulin binding to ATP extractable brush-border membrane myosin I and 1,25-(OH) 2D 3 stimulated changes of calcium uptake across the brush-border membrane.