Tamio Hirabayashi

Two-dimensional gel electrophoresis of chicken skeletal muscle proteins with agarose gels in the first dimension

Abstract An improved method of two-dimensional gel electrophoresis is described. The method is specifically developed for preparing a “protein map” of chicken skeletal muscle, and is found to be applicable to the analysis of most protein constituents including high molecular ones, such as myosin heavy chain, without using any detergents in the first dimension. Omission of detergents from the focusing medium results in two advantages. (i) The first-dimension isoelectric focusing pattern can be recorded by taking a photograph of the gel prior to the second-dimension electrophoresis, so that even a close doublet band in the first dimension, which forms one spot in the second dimension, can be found heterogeneous in component by examining the first-dimension pattern of the same gel. (ii) Since peptides of relatively large molecular weights can be analyzed by first-dimension isoelectric focusing, complex formation between polypeptides with different isoelectric points is demonstrable. For example, troponin T, troponin I, and troponin C are found by two-dimensional gel electrophoresis to form a complex in a 4 m urea solution, and so are troponin I and troponin C in a 5 m urea solution.

Developmental change of protein constituents in chicken gizzards.

Developmental change of protein constituents of chick gizzard smooth muscle was described by the fluorescent antibody technique and two-dimensional polyacrylamide gel electrophoresis. Myosin heavy chain, tropomyosin, and desmin were immunohistologically detected in 5-day-old gizzard primordia, but myoglobin was detected after 19 days of incubation. Two-dimensional polyacrylamide gel electrophoresis revealed that most structural proteins including beta- and gamma-actin are synthesized almost simultaneously in the primordium, and accumulate in three patterns by which the proteins examined are classified: (1) gradually increasing protein (gamma-actin, tropomyosin, desmin), (2) abruptly increasing protein at a certain stage (myosin, myoglobin), (3) decreasing or constantly kept protein (tubulin, beta-actin). Based on the quantitative analysis of protein constituents, the nature of regulatory system of protein synthesis in smooth muscle and the possible functional difference between beta- and gamma-actin are discussed.

Tetrahymena calmodulin: Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in tetrahymena

Abstract A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlonys immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium , but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti- Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.

Isolation and electrophoretic properties of a calcium-binding protein from the ciliate Tetrahymena pyriformis

Abstract A calcium-binding protein (TCBP) was isolated from the ciliate Tetrahymena pyriformis by a procedure which included 8 M urea extraction of the cell homogenate or boiling water extraction of acetone-dried powder of the cell, ammonium sulfate precipitation (60–80% saturation) and preparative electrophoresis on alkaline polyacrylamide gel. The TCBP has a molecular weight of 14,000 daltons and an isoelectric point of pH 4.0 and is easily detected in crude cell extracts by the change of electrophoretic mobility depending on Ca ion concentration. By equilibrium dialysis TCBP was found to bind to 2 moles of calcium per mole of the protein at a free calcium concentration of 10 −4 M. On alkaline polyacrylamide gel, the TCBP interacts with rabbit skeletal muscle troponin I and with some material(s) of Tetrahymena origin to form Ca 2+ -dependent complexes.

Expression pattern of skeletal muscle troponin T isoforms is fixed in cell lineage

The expression of fast-muscle-type troponin T isoforms in chicken skeletal muscles was studied by two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting. According to the pattern of troponin T isoform expression, chicken fast muscle was classified into two groups: One group expressed breast-fast-muscle-type troponin T in addition to leg-fast-muscle-type troponin T, the other expressed only leg-fast-muscle-type troponin T. To the former group belong breast and wing fast muscles and some of the back fast muscles, and to the latter group belong the fast muscles in leg, abdomen, and neck. Transplantation of breast muscle into leg was performed in order to change the physical environment and to investigate the mechanism of isoform expression. Histological observation of the transplant revealed severe degeneration of muscle cells, followed by differentiation of myoblasts in which breast-muscle-type troponin T was eventually expressed. The results showed that the pattern of troponin T isoform expression is primarily fixed in the cell lineage, although nerves modulate it.

Coordinate accumulation of troponin subunits in chicken breast muscle

The accumulation of troponin subunits in developing chicken breast muscle was determined by two-dimensional gel electrophoresis and an image analyzing system. Many troponin T isoforms, including those hidden behind creatine kinase, were detected on the two-dimensional pattern by the addition of 6 M urea in the second dimension. These troponin T isoforms were classified into four types by developmental order, isoelectric point, and molecular weight: leg-muscle type (L), neonatal breast-muscle type (BN), young chicken breast-muscle type (BC), and adult breast-muscle type (BA). The L-, BN-, and BC-type troponin Ts were transiently expressed at specific developmental stages. Quantitative analysis of two-dimensional patterns of troponin subunits including troponin I and troponin C showed moderate coordination in accumulation among the three subunits throughout postnatal development, when the total amount of all isoforms of troponin T was taken into account.

Calcitonin Gene‐Related Peptide‐Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization

Abstract: Calcitonin gene‐related peptide (CGRP)‐binding sites were solubilized, using digitonin, from the porcine spinal cord, atria, and coronary arteries. The specific binding of 125I‐human α‐CGRP to the solubilized binding sites was inhibited by human α‐ and β‐CGRP and by rat α‐CGRP, but not by angiotensin II or human calcitonin. Scatchard plot analysis of saturation gave the same KD value for CGRP in the crude membrane fractions of the tissues examined. The affinity of CGRP to the binding sites was decreased by solubilization in the atria and coronary arteries, but not in the spinal cord. Affinity labeling followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed distinct molecular sizes of the specific binding sites among the tissues; 70K for the spinal cord, 70K and 90K for the coronary arteries, and 70K and 120K for the atria. These results indicate that the molecular characteristics of the specific binding sites of CGRP in the cardiovascular system are distinct from those in the central nervous system.

A new fiber-forming protein from Tetrahymena pyriformis.

Abstract A new fiber-forming protein was isolated from the acetone powder of Tetrahymena pyriformis by co-precipitating with skeletal muscle myosin while trials were made to find actin or actin-like protein in Tetrahymena . It has a molecular weight of 38000 D and forms a tetramer (140000 D, 9 S) in physiological conditions. Its isoelectric point (pH 6.7), amino acid composition and antigenic determinant(s) differ significantly from those of non-muscle actin and skeletal muscle actin. It does not undergo G-F conversion while actin does, and does not activate Mg 2+ -ATPase of skeletal muscle myosin. The protein localizes in the oral apparatus and division furrow as revealed by fluorescent antibody method. The protein can be assembled into 14-nm filaments in a reassembly buffer. The in vitro filaments appear to correspond to some filaments included in the oral apparatus and the contractile ring. The fiber-forming protein from Tetrahymena may play important roles in cell motility including cell division.

Detection of dystrophin on two-dimensional gel electrophoresis.

A protein with MW approximately 350 k daltons and pI approximately 5.5, which was deleted in the dystrophic mouse (C57BL/10ScSn-mdx), was detected on two-dimensional gel electrophoresis with silver staining. Deletion of this protein was uniformly observed in the dystrophic mouse extensor digitus longus, soleus and cardiac muscle. This protein specifically reacted with the monoclonal antibody against the chemically synthesized N-terminal fragment of human dystrophin. The protein reacting with this monoclonal antibody was also detected in rabbit back-muscle, rat extensor digitus longus and human skeletal muscle at the same position as the mouse muscle protein, on the two-dimensional gel electrophoresis. Our results show that dystrophin is solubilized in 8M guanidine HCl and that the modified two-dimensional gel electrophoresis can be applied to separate dystrophin.

Comparison of protein constituents between atria and ventricles from various vertebrates by two-dimensional gel electrophoresis

1. Protein constituents of cardiac muscles of 23 species were examined by two-dimensional gel electrophoresis in order to find the difference in protein components between atria and ventricles. 2. Protein compositions of atria were similar to those of ventricles, however, differences were found in myosin and some other proteins in most species. 3. A major protein with molecular weights of 12,000-15,000 daltons was distributed only in atria from mammals. 4. The atrioventricular difference suggested that the ventricular tissue was more specialized in mammals than in birds, as compared with the atrial tissue.