Large-area hard magnetic L10-FePt nanopatterns by nanoimprint lithography.

Large-area hard magnetic L1(0)-FePt nanopatterns with out-of-plane texture were fabricated by using a top-down approach. For the fabrication process, ultraviolet nanoimprint lithography (UV-NIL) in combination with inductively coupled plasma reactive Ar-ion etching was used. By this technique a continuous L1(0)-Fe(51)Pt(49) film was nanostructured into a regular arrangement of nanodots over an area of 4 mm(2). The dot dimension and distribution was specified by the stamp, resulting in a dot size of 60 nm and a periodicity of 150 nm. For the large-scale L1(0)-FePt nanopatterns, huge coercivities up to 4.31 T could be achieved. By means of magnetic force microscopy it could be verified that the nanodots were magnetically decoupled from each other and occurred in the single-domain state with perpendicular magnetization.

Forest production efficiency increases with growth temperature.

Forest production efficiency (FPE) metric describes how efficiently the assimilated carbon is partitioned into plants organs (biomass production, BP) or-more generally-for the production of organic matter (net primary production, NPP). We present a global analysis of the relationship of FPE to stand-age and climate, based on a large compilation of data on gross primary production and either BP or NPP. FPE is important for both forest production and atmospheric carbon dioxide uptake. We find that FPE increases with absolute latitude, precipitation and (all else equal) with temperature. Earlier findings-FPE declining with age-are also supported by this analysis. However, the temperature effect is opposite to what would be expected based on the short-term physiological response of respiration rates to temperature, implying a top-down regulation of carbon loss, perhaps reflecting the higher carbon costs of nutrient acquisition in colder climates. Current ecosystem models do not reproduce this phenomenon. They consistently predict lower FPE in warmer climates, and are therefore likely to overestimate carbon losses in a warming climate.

Morphology of rigor--shortened bovine muscle and the effect of trypsin on pre- and postrigor myofibrils.

Bovine semitendinosus muscles were sampled immediately after death, after 24 hr postmortem with storage at 2 degrees , 16 degrees , or 37 degrees C, and after 312 hr postmortem with storage at 2 degrees and 16 degrees C. A biopsy technique was used to prevent shortening during glutaraldehyde fixation. Postfixation in osmium tetroxide was followed by embedding in an Epon-Araldite mixture. Bovine muscle was supercontracted after 24 hr storage at 27deg; but was only slightly contracted after storage at 16 degrees for 24 hr. Muscle held at 37 degrees for 24 hr was slightly less supercontracted than the 2 degrees muscle. Striking similarities existed between muscles stored at 16 degrees and at 2 degrees C for 312 hr. Both were slightly shortened with narrowed I bands and an area of increased density, probably due to overlap of thin filaments in the middle of the A band. Postmortem shortening was accompanied by banding-pattern changes similar to those predicted for contracting muscle by Huxley and Hanson's sliding filament model. Treatment of myofibrils with 0.05% trypsin resulted in a rapid loss of Z lines and, in supercontracted myofibrils, caused a return of the banding pattern of resting muscle.

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa.

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb-IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.

Sythesis of tropomyosin in cultures of differentiating muscle cells.

The accumulation of tropomyosin in cultures of differentiating muscle cells was quantitatively measured. Tropomyosin was isolated from cultured cells during and after myoblast fusion; both alpha- and beta-subunits were present in myotube cultures. During fusion small amounts of tropomyosin were detectable, but, as fusion approached a maximum, tropomyosin accumulation began to increase. The increased synthesis of tropomyosin after the initiation of muscle cell fusion is consistent with the increased synthesis of other proteins characteristic of muscle, including myosin.

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle.

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca(2+) and 5 nM Mg(2+) for 9 hr at 37 degrees C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca(2+) and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37 degrees C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca(2+) and 5 mM Mg(2+) in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca(2+) at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0-7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca(2+) levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca(2+) in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.

Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active.

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle.

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.

Global patterns of phosphatase activity in natural soils.

Soil phosphatase levels strongly control the biotic pathways of phosphorus (P), an essential element for life, which is often limiting in terrestrial ecosystems. We investigated the influence of climatic and soil traits on phosphatase activity in terrestrial systems using metadata analysis from published studies. This is the first analysis of global measurements of phosphatase in natural soils. Our results suggest that organic P (Porg), rather than available P, is the most important P fraction in predicting phosphatase activity. Structural equation modeling using soil total nitrogen (TN), mean annual precipitation, mean annual temperature, thermal amplitude and total soil carbon as most available predictor variables explained up to 50% of the spatial variance in phosphatase activity. In this analysis, Porg could not be tested and among the rest of available variables, TN was the most important factor explaining the observed spatial gradients in phosphatase activity. On the other hand, phosphatase activity was also found to be associated with climatic conditions and soil type across different biomes worldwide. The close association among different predictors like Porg, TN and precipitation suggest that P recycling is driven by a broad scale pattern of ecosystem productivity capacity.

Effect of alpha-actinin on actin structure. Actin ATPase activity.

Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers.

cAMP responsiveness of the bovine calpastatin gene promoter.

Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were located within -102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt -76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt -1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt -76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.

Activation of coagulation factor V by calcium-dependent proteinase.

Factor V is a key coagulation cofactor, regulating the rate of Factor Xa-catalyzed prothrombin conversion. Activation of Factor V markedly accelerates coagulation. This study describes a new class of Factor V activators, sulfhydryl proteinases. Of the enzymes studied, calcium-dependent proteinase was the most effective activator. Activation of Factor V by this enzyme was associated with cleavage of 125I-labeled Factor V to peptides distinct from those generated by previously described activators. Calcium-dependent proteinase-activated Factor Va peptides with molecular weights of 114,000 and 93,000 bound both to Factor Xa and to cultured endothelial cells. Calcium-dependent proteinase was identified in vascular endothelial cells, a tissue that also synthesizes Factor V. These findings suggest a previously unknown mechanism for cellular regulation of coagulation.

N- and C-terminal amino acids of purified alpha-actinin.

Highly purified bovine cardiac alpha-actinin is obtained by successive chromatography on DEAE-cellulose and hydroxyapatite of a crude fraction obtained by salting out low ionic strength extracts of bovine cardiac muscle between 0 and 30% ammonium sulfate saturation. Hydroxyapatite chromatography removes a 43 000-dalton polypeptide chain that is difficult to remove by successive DEAE-cellulose columns. Removal of all 43 000-dalton material by hydroxyapatite chromatography is accompanied by disappearance of a very small 9 to 10 S boundary in analytical ultracentrifuge diagrams of DEAE-cellulose-purified 6.2S alpha-actinin. Approximately 95% of the protein in DEAE-cellulose and hydroxyapatite-purified alpha-actinin is the 100 000-dalton alpha-actinin polypeptide as estimated by SDS-polyacrylamide gel electrophoresis. Purified bovine cardiac, porcine skeletal, chicken gizzard, and chicken breast alpha-actinins all contain leucine as the C-terminal amino acid of both polypeptide chains in the alpha-actinin molecule. Bovine cardiac and porcine skeletal alpha-actinins contain arginine as the amino acid penultimate to C-terminal leucine. None of the four different alpha-actinins studied had a N-terminal amino group available for reaction with dansyl chloride, but all four alpha-actinins contained 1.6 to 1.8 acetate residues per molecule (200 000 daltons) of alpha-actinin. It seems likely that the N-terminal amino groups of both polypeptide chains in these four alpha-actinins are acetylated. A peptide having the composition N-Ac-Asp2-Glu4 was isolated from a proteolytic digest of bovine cardiac alpha-actinin. alpha-Actinin seems to be a conserved protein molecule found in many different motile systems.

Effect of alpha-actinin on actin structure: viscosity studies.

The effect of ATP on ability of alpha-actinin to increase viscosity of F-actin was measured in three different solutions: 100 mM KCl; 100 mM KCl/l mM Mg2+; and Mg2+ alone at concentrations of 1-6 mM. When ATP and Mg2+ are added at equimolar ratios or at added [ATP] to added [Mg2+] greater than equimolar, alpha-actinin has no effect on F-actin viscosity in the absence of KCl. ATP decreases viscosity of alpha-actinin/F-actin mixtures by 20% even in the presence of KCl, evidently because ATP affects the alpha-actinin-F-actin interaction. Molar ratios of 1 alpha-actinin to 49 actins increase specific viscosity of F-actin approx. 2-fold at 37 degrees C in the presence of 1 mM ATP, so ATP does not prevent the alpha-actinin-F-actin interaction.

Cross-linking and proteolysis in Ca2(+)-treated lens homogenates.

It was previously shown (Lorand et al. (1985) Biochemistry 24, 1525) that treatment of lens homogenate with Ca2+ produces two sets of changes which are catalyzed by intrinsic enzymes of the lens and which can be readily seen by alterations in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of proteins. With the aid of differential inhibitors of the two reactions (e.g., dansylcadaverine and leupeptin) it was possible to distinguish the transglutaminase-dependent cross-linking of proteins from the proteolytic degradative phenomena. We have now shown that the proteins which are affected by the two processes can be compartmentalized differentially by centrifuging the lens homogenate after exposure to Ca2+. The dimeric and oligomeric beta-crystallin products of transglutaminase-mediated cross-linking are most clearly visible in the soluble supernatant, whereas the proteolytically susceptible proteins--possibly structural in nature, including vimentin--are predominantly present in the pellet. We have found a compound, 2-[3-(diallylamino)propionyl]benzothiophene, which, by virtue of acting as a noncompetitive inhibitor of transglutaminase as well as of calpains I and II, effectively blocked both the cross-linking seen in the supernatant and the proteolysis seen in the pellet fraction, though perhaps with somewhat different sensitivities.

Comparison of the autolyzed and unautolyzed forms of mu- and m-calpain from bovine skeletal muscle.

Bovine skeletal muscle mu- and m-calpain autolyze when incubated with Ca2+. During the first 30 to 300 s, autolysis: (1) has little effect on the specific proteolytic activity of either mu- or m-calpain when assayed at 5 mM Ca2+; and (2) produces two new proteolytically active forms of calpain in addition to the original mu- and m-calpain. The four proteolytically active forms of calpain are: (1) autolyzed mu-calpain, having polypeptide subunits of 76 and 18 kDa and requiring 0.60 microM Ca2+ for half-maximal activity; (2) mu-calpain with 80- and 28-kDa subunits and requiring 7.1 microM Ca2+ for half-maximal activity; (3) autolyzed m-calpain with 78- and 18-kDa subunits and requiring 180 microM Ca2+ for half-maximal activity; and (4) m-calpain with 80- and 28-kDa subunits and requiring 1000 microM Ca2+ for half-maximal activity. All four forms of the calpains have similar pH optima (7.4 to 7.6) and almost identical circular dichroism spectra in the far ultraviolet (all four have little secondary structure with 26-30% alpha-helix and less than 10% beta-sheet structure). Autolyzed mu- and unautolyzed mu-calpain are fully activated proteolytically by Mn2+ with activity starting at 125 microM Mn2+. Autolyzed m-calpain is also activated by Mn2+ up to 80% of the maximum proteolytic activity obtained with Ca2+; Mn2+ activation begins at 320 microM Mn2+. Unautolyzed m-calpain has only 6 to 8% as much activity in the presence of Mn2+ as it does in the presence of Ca2+. Autolysis increases the axial ratios of the calpains from 3.5 to 4.6 for mu-calpain and from 3.7 to 5.0 for m-calpain (assuming 20% hydration). The estimated length of the calpain molecules increases by 13% upon autolysis from 73 to 84 A for mu-calpain and from 76 to 90 A for m-calpain (assuming 20% hydration). The autolyzed calpains elute after their unautolyzed counterparts off a DEAE-ion exchange column. Because autolyzed forms of the calpains are not found in DEAE elution profiles of cell extracts, bovine skeletal muscle cells must contain very little (less than 5% of total calpain) or none of the autolyzed form of the calpains.

Chicken skeletal muscle has three Ca2+-dependent proteinases.

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.

Effect of trypsin on rabbit skeletal muscle alpha-actinin.

5 min of tryptic digestion of purified rabbit skeletal alpha-actinin decreases by approximately 75% the ability of alpha-actinin to cross-link F-actin filaments as measured viscometrically at 27 degrees C, but has little effect on the sedimentation coefficient of alpha actinin at 20 degrees C or an alpha-actinin's ability to increase the Mg2+-modified ATPase activity and rate of turbidity increase of reconstituted actomyosin suspensions. Twenty to sixty min of trypsin treatment reduces the sedimentation coefficient of alpha-actinin and destroys much of alpha-actinin's ability to increase the MG2+-modified ATPase and rate of turbidity increase of reconstituted actomyosin suspensions. Therefore, the ability of alpha-actinin to increase the rate of in vitro measures of muscle contraction may not result directly from alpha-actinin's ability to cross-link F-actin filaments. Trypsin does not split alpha-actinin into large fragments as it does myosin. Previous studies have shown that 35 to 65% of total tryptic-susceptible peptide bonds in alpha-actinin are split after 60 min of incubation with trypsin and that 30% of these bonds split in 60 min are cleaved during the first 5 min in a rapid reaction. That splitting of this group of peptide bonds has little effect on the sedimentation coefficient of alpha-actinin indicates that these bonds are located in a region of the alpha-actinin molecule where noncovalent forces are strong enough to maintain conformation of the native alpha-actinin molecule even after these bonds have been split. This ostensible segregation of alpha-actinin's ability to cross-link F-actin filaments from its ability to increase rate of in vitro assays of contraction by tryptic digestion may suggest that alpha-actinin could have at least two different physiological roles: (1) to bind actin filaments to each other or to basal structures, and (2) to enhance the effectiveness of actin in supporting movement.

Effect of alpha-actinin on actin structure. Release of bound nucleotide.

We have examined the alpha-actinin-F-actin interaction by measuring the effect of highly purified alpha-actinin on bound nucleotide exchange in F-actin. Exchange was followed by measuring the release of actin-bound [14C]ADP in the presence of ATP using an ultrafiltration technique. Alpha-Actinin increases by about 60 to 70% the rate of release of F-actin bound nucleotide when incubated for 1 h in the presence of 1 mM ATP/1 mM MgCl2/0.05 mM CaCl2/0.5 mM dithioerythritol/100 mM KCI/20 mM Tris-acetate, pH 7.5, at 37 degrees C. The ability of alpha-actinin to enhance nucleotide exchange was maximal when alpha-actinin was added at a level near 10% of actin present by weight (molar ratio of 1 alpha-actinin to 49 actin monomers). The potentiating effect of alpha-actinin on the nucleotide exchange rate of F-actin was not highly related to the Mg2+: ATP ratio present in the incubation mixture. Alpha-actinin also increased the rate of bound nucleotide exchange of f-actin was present in a reconstituted actomyosin suspension. The results are consistent with th possibility that one alpha-actinin can affect the structure of multiple actin monomers present in an actin filament.

Disproportionate accumulation of myosin and tropomyosin in cultured muscle cells.

Accumulation of two major myofibrillar proteins, myosin and tropomyosin, was monitored in differentiating skeletal muscle cultures. The tropomyosin subunit to myosin heavy chain accumulation rate ratio was more than twice the stoichiometric ratio of tropomyosin subunit to myosin heavy chain in mature skeletal muscle myofibrils and crude myofibrils from cultured muscle cells. Electron microscopy revealed normal patterns of myofibril assembly in these muscle cultures. Therefore, the observed disproportionate accumulation of tropomyosin and myosin heavy chain may be reflecting normal cellular conditions for de novo myofbril assembly.