Luciano Dalla Libera

Myosin light and heavy chains in muscle regenerating in absence of the nerve: Transient appearance of the embryonic light chain

We examined myosin of fast and slow skeletal rat muscles regenerating after ischemia and bupivacaine injection in denervated limbs. Four days after injury two-dimensional gel electrophoresis revealed the presence of the embryonic light chain in the myosin isolated from the portion of muscle showing a homogeneous population of new small fibers by histological examination. Two weeks after injury this subunit was absent, whereas the two light chains, LC1F and LC2F, became prominent. One month after injury the still denervated soleus muscle maintained this light chain pattern. Gel electrophoresis in native condition of the myosin and peptide mapping of electrophoretically purified heavy chains confirmed that the muscle regenerating in absence of the nerve accumulated a myosin that had the general features of a fast, not slow, myosin but contained definite differences from the former.

Myosin light and heavy chains in rat gastrocnemius and diaphragm muscles after chronic denervation or reinnervation

Abstract Myosin of chronically denervated and reinnervated rat muscles was studied. The two-dimensional gel electrophoretic pattern of light chains and the tryptic mapping of the native molecule show that almost only fast-type myosin was present in 6-month denervated hemidiaphragm and gastrocnemius muscles, whereas reinnervated myosins were mixed in the respective controls. The chymotryptic peptide mapping under denaturing conditions of electrophoretically purified myosin heavy chains confirmed that the observed changes concerned not only light chains but also the heavy subunits of the myosin. The results of reinnervation experiments allow one to conclude that the selective maintenance of fast-type myosin in chronically denervated muscles cannot be attributed to minimal reinnervation events.

Immunological and biochemical evidence for atrial-like isomyosin in thyrotoxic rabbit ventricle

Immunological, structural and enzymatic characteristics of atrial and ventricular myosin from euthyroid rabbits were analyzed and compared with ventricular myosin from hyperthyroid animals. (1) Specific antibodies against bovine atrial myosin were found to react selectively in double-immunodiffusion and enzyme immunoassay with both rabbit atrial myosin and ventricular myosin from thyroxine-treated animals. These specific anti-bovine atrial myosin antibodies reacted with the heavy chains of thyrotoxic ventricular myosin when examined by enzyme immunoassay combined with SDS-polyacrylamide gel electrophoresis. (2) In one-dimensional SDS-polyacrylamide gel electrophoresis, no difference could be demonstrated in the light chain pattern of ventricular myosin from euthyroid and hyperthyroid rabbit hearts. One-dimensional analysis of myosin heavy chains after chymotryptic digestion in the presence of SDS showed significant differences between the two ventricular myosins. Also, the peptide maps from atrial myosin resembled the pattern of peptides found with ventricular heavy chains from hyperthyroid rabbits. The steady state rate, the alkali stability and the pH sensitivity of Ca2+-ATPase activity of thyrotoxic ventricular myosin were very similar to those of atrial myosin. (3) These results provide direct immunochemical and biochemical evidence for the existence of an atrial-like isomyosin in thyrotoxic rabbit ventricles.

DIFFERENTIAL DISTRIBUTION OF TROPOMYOSIN SUBUNITS IN FAST AND SLOW RAT MUSCLES AND ITS CHANGES IN LONG-TERM DENERVATED HEMIDIAPHRAGM

Skeletal tropomyosin, a rod-shaped protein which lies in the grooves of the double-stranded F-actin filament, is involved in the calcium regulatory system of muscle contraction [ 11. The native molecule is a dimer in which the two subunits (Y (34 000 MJ and B (36 000 Mr) are assembled as cm, Pf.3 or 43; in the slow and fast mammalian muscles the ratio of molar amount of OL and fl subunits of tropomyosin differs, the P-subunit being more represented in the slow type and during the development [2,3 1. The distribution of tropomyosin subunits has been studied in several mammalian species by immunohistochemical studies and electrophoretic analysis of extensively purified tropomyosin. Results have been collected by two-dimensional electrophoresis [6]. The position of the tropomyosin subunits have been identified in a two-dimensional electrophoretogram of crude myofibrils from rat muscle, using tropomyosin purified by standard techniques [7]. However, their relative proportion in the fast and slow muscles has not been determined. We have performed the two-dimensional electrophoresis of actomyosin from normal and long-term denervated rat muscles. The tropomyosin subunit pattern was thus compared with the myosin light chain pattern in the same preparation. The two-dimensional electrophoresis shows that adult rat soleus, a predominant slow type muscle, contains almost exclusively thee-subunit of tropomyosin, while adult fast and immature muscles contain both CX- and &subunits in about the same proportion. This is a further evidence that tropomyosin subunit ratio differs in fast and slow skeletal muscles, even though with a distribution peculiar to each mammalian species. The analysis of tropomyosin subunits and myosin light chains shows that

Microplate enzyme-linked immunosorbent assay in the study of the structural relationship between myosin light chains

A microplate enzyme-linked immunosorbent assay (microELISA) for the study of immunochemical relationships between rabbit myosin light chains is described. Purified individual fast-muscle myosin light chains (LC1F, LC2F and LC3F) and their respective antisera, obtained in chicken, were used. Optimal conditions for antigen concentration, antiserum dilution, substrate concentration, incubation time and reproducibility with time were established. The observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin confirm and extend previous results obtained by other authors using radioimmunoassay procedures. It was concluded that microELIAS may be successfully employed also to the study of macromolecule cross-reactivities.

Smooth muscle myosin light chain kinase: Rapid purification by anion exchange high-performance liquid chromatography

A rapid procedure for the purification of myosin light chain kinase present in chicken gizzard smooth muscle using anion exchange high-performance liquid chromatography is described. The procedure allows preparation of microgram amounts of the protein directly from the extract of gizzard myofibrils and then is suitable for the study of myosin light chain kinase in small muscles. The protein was judged to be greater than 95% pure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme retains its activity since it catalyzes the calcium-calmodulin-dependent phosphorylation of the 20,000-Da myosin light chain.

Heterogeneity of smooth muscle myosin light chain kinase is revealed by anion-exchange high-performance liquid chromatography

When smooth muscle myosin light chain kinase, purified by standard procedures from chicken gizzard smooth muscle, was applied to an anion-exchange high-performance liquid chromatographic column, three well resolved peaks were obtained. Each peak contained a single protein whose electrophoretic mobility corresponded to that of MLCK. However each enzyme was characterized by a different specific activity. Peptide mapping experiments were unable to demonstrate different proteolytic patterns for the three proteins. Treatment of myosin light chain kinase with alkaline phosphatase, prior to ion chromatography, resulted in a change of elution profile. These experiments suggest that myosin light chain kinase could exist in three forms characterized by a different degree of phosphorylation.

Biochemical characterization of ventricular myosin from spontaneously hypertensive turkeys

Several stimuli are able to alter the synthesis of cardiac myosin isoenzymes. Particularly in the rat a shift toward a low-ATPase isomyosin is generally observed during development and in cardiac hypertrophy due to pressure overload. On the contrary in spontaneously hypertensive turkeys both ageing and the increasing degree of cardiac hypertrophy are accompanied by a different behaviour of ventricular myosin. In fact in a previous study we have shown that the Ca2+-activated ATPase activity of ventricular myosin increases about three folds from young normotensive to old hypertensive animals. Accordingly the peptide pattern obtained after chymotryptic digestion of myosin showed that some peptides, which are not evident or barely discernible in young animals, are present in the adult ones. In this study we compare the ventricular myosin from young normotensive and adult hypertensive turkeys with atrial myosin. The results obtained suggest that in the ventricles of hypertensive turkeys the synthesis of an isomyosin with biochemical properties close to those of atrial myosin occurs.