A simple and rapid method to remove light chain phosphatase from chicken gizzard myosin

A simple and rapid method to remove myosin light chain phosphatase from gizzard myosin using hydroxyapatite chromatography was developed. Stable phosphorylated myosin without light chain phosphatase and kinase was also prepared by the same procedure. A convenient method using the urea gel system to detect minor contamination by phosphatase of myosin is also described.     

Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schr?er, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.     

A simple method of preparing myosin from porcine aortae

A simple method for preparing myosin from porcine aortae is described. The procedure yields a highly pure myosin devoid of any obvious contaminants, such as tropomyosin and actin. The proteolysis of the heavy chain, frequently observed in the earlier procedures, is greatly minimized by the use of several protease inhibitors and by reducing the purification time. The procedure therefore can be used to isolate and purify myosin from porcine aortae for studies where the purity and integrity of the heavy chain are highly desirable.     

Photocrosslinking of calmodulin and/or actin to chicken gizzard caldesmon

The two sulfhydryl groups of chicken gizzard caldesmon were specifically labeled with a photoreactive crosslinker, benzophenone-maleimide, to study its interactions with calmodulin and/or actin. When incubated with F-actin caldesmon crosslinks to a single actin monomer; it can, however, crosslink to up to two calmodulin molecules in the presence, but not in the absence, of Ca2+. Thus caldesmon may have two calmodulin-binding sites, each containing, or being near, one of the two thiol residues. One of these two sites may also be adjacent to the actin-binding site. A calmodulin-binding fragment of caldesmon resulting from cyanogen bromide digestion crosslinks to a single calmodulin molecule, also in a Ca2+-dependent manner. Crosslinking of calmodulin to caldesmon does not prevent the latter from binding F-actin, suggesting that calmodulin and actin do not compete with each other for the same binding site(s) on the caldesmon molecule.     

A simple method for preparing meiotic chromosomes from mammalian testis

Freshly removed testis tubules are treated with hypotonic citrate solution and fixed in 31 acetic alcohol without undue rupture. The material is transferred to 60% acetic acid and the resulting suspension is air-dried onto warm slides using a micropipette. Finally the slides are stained with lacto-acetic orcein. — Provided suitable storage is made, slides need not be prepared in the same week as fixation and this may facilitate the collection of material during field study.     

Glycosaminoglycans from chicken muscular stomach or gizzard

Glycosaminoglycans (GAGs) were prepared from the muscular stomach or gizzard of the chicken. The content of GAGs on a dry weight basis contains 0.4 wt.% a typical value observed for a muscle tissue. The major GAG components were chondroitin-6-sulfate and chondroitin-4-sulfate (~64 %) of molecular weight 21–22 kDa. Hyaluronan (~24 %) had a molecular weight 120 kDa. Smaller amounts (12 %) of heparan sulfate was also present which was made of more highly sulfated chains of molecular weight of 21-22 kDa and a less sulfated low molecular weight (< 10 kDa) heterogeneous partially degraded heparan sulfate. Chicken gizzard represents an inexpensive and readily available source of muscle tissue-derived GAGs.     

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

Properties of talin from chicken gizzard smooth muscle

This paper describes the structural and biochemical characterization of talin, a protein localized to various cellular sites where bundles of actin filaments attach to the plasma membrane. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 225,000 +/- 5,000 daltons. Hydrodynamic measurements at protein concentrations less than 0.72 mg/ml indicate a monomeric protein with a native molecular mass of 213,000 +/- 15,000 daltons. Sedimentation equilibrium experiments indicate self-association at protein concentrations of 0.72 mg/ml and higher. The data suggest that this self-association is a simple monomer:dimer equilibrium over the range of concentrations observed. At low protein concentrations where talin is a monomer, the Stokes radius and sedimentation coefficient vary with ionic strength. Under low ionic strength conditions (5-20 mM NaCl), talin has a Stokes radius of 6.5 nm and a sedimentation value of 9.4, suggesting an asymmetric globular molecule; whereas under high ionic strength conditions (200 mM NaCl), the Stokes radius increases to 7.7 nm and the sedimentation coefficient decreases to 8.8, suggesting a more elongated protein. This conformation change is confirmed by electron microscopy which reveals a more globular protein at low ionic strength which unfolds to become an elongated flexible molecule as the ionic strength is increased to physiological and higher levels. The amino acid composition of talin indicates a low level of aromatic residues, consistent with its relatively low extinction coefficient, talin has an isoelectric point between pH 6.7 and 6.8 based on isoelectric focusing. The detailed purification of talin is described.     

A simple method of preparing diverse neoagaro-oligosaccharides with beta-agarase

In order to prepare pure and well-defined oligosaccharides from agarose in a rapid and simple manner, an enzymatic degradation method was developed, which includes degradation with either recombinant beta-agarase (EC 3.2.1.81) AgaA or AgaB and gel permeation chromatography. Agarose was degraded with AgaA at the optimized conditions, yielding 47% and 45% of neoagarotetraose and neoagarohexaose, respectively. These neoagaro-oligosaccharides were conveniently separated by consecutive column chromatography on Bio-Gel P2 or P6 and were identified by FACE. The structure of these neoagaro-oligosaccharides was confirmed by MALDI-TOF MS and (13)C NMR spectroscopy.     

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.     

A new method of preparing monocyte suspensions from human whole blood

A method of isolating monocytes from human whole blood is described. The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens. Counterflow centrifugation is not involved. The final monocyte suspension is free of platelets. The contaminating cells are predominantly lymphocytes. As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.     

Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery

Rhodamine-labeled actin microinjected into living embryonic chicken gizzard cells became associated with its characteristic cytoskeletal structures. In these domains the translational diffusion coefficients (D) of rh-actin were determined in vivo by fluorescence photobleaching recovery (FPR) measurements. Two classes of actin molecules with respect to its mobilities were detected: rh-actin with a half-time of recovery of 5-10 min in stress fibers and focal contacts (immobile on the time-scale of FPR measurements) and rh-actin with D = 2-3 X 10(-9) cm2/sec in the cytoplasm and leading lamellae. The slow recovery on stress fibers exhibited similar kinetics whether a short segment or the entire structure were photobleached, indicating that recovery occurs predominantly by exchange with the surrounding diffusable actin. We propose that a steady-state equilibrium between the soluble and cytoskeletal pool of actin exists in living cells.     

Stability properties of oxymyoglobin from chicken gizzard smooth muscle

1. Oxymyoglobin (MbO2) was isolated directly from the smooth muscle of chicken gizzard and was examined for its spectral and stability properties. 2. When compared with sperm whale MbO2 as a reference, chicken gizzard MbO2 was found to be much more susceptible to autoxidation. Its pH-dependence was therefore analyzed in terms of an "acid-catalyzed three-state model". 3. The complete amino acid sequence of the myoglobin was also determined. Its hydropathy profile revealed that the region corresponding to the distal side of the heme iron appears to be less hydrophobic.     

Two chymotrypsin-susceptible sites of myosin rod from chicken gizzard

The rod prepared from chicken gizzard myosin has been found to have two sites sensitive to limited digestion with chymotrypsin; these sites were located at a subfragment 2/light meromyosin junction (site 1), and at a site 10 kDa remote from either C-terminal or N-terminal of light meromyosin (site 2). The site 1 was more sensitive to the digestion than the site 2. The cleavage at site 2 of the light meromyosin yielded a 74-kDa fragment that was soluble in a low ionic strength solution, contrary to the insolubility of the parent light meromyosin in the same solution. Studies on the effects of MgCl2, ATP and pH on the susceptibilities of these sites to chymotrypsin have given following results. (a) Millimolar concentrations of MgCl2 protected site 1 and site 2 from the chymotryptic cleavage. (b) The cleavage at site 1 of myosin rod in the low salt solution free of Mg2+ at pH 7.0 and pH 8.5, was not affected by the presence of 5 mM ATP. However, MgCl2-induced protection of site 1 was relieved by addition of ATP. On the other hand, the cleavage at site 2 was stimulated by addition of ATP, irrespective of the presence or absence of MgCl2. (c) The alkaline condition of pH 8.5 was more favorable for the chymotryptic cleavages at both site 1 and site 2 than the neutral condition of pH 7.0. These results suggest that myosin rod contains two flexible regions, the structures of which are influenced by such an ambient factor as MgCl2, ATP or pH.     

A simple method of preparing split-thickness skin grafts for meshing

A simple and rapid technique of mounting a split-thickness skin graft on the Tanner dermacarrier is presented. Initially, petroleum jelly is applied onto the grooved surface of the dermacarrier. The skin graft is not removed from the donor site; instead, the skin graft knife is withdrawn backwards, allowing the graft to slip back, smoothly and evenly, onto the donor site through the knife. The dermacarrier, with the petroleum jelly, is laid onto the graft. The graft adheres to the dermacarrier because of the jelly. No further maneuver is necessary prior to meshing. With this technique it is impossible to apply the graft cut-side up on the recipient site.