Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.     

Phosphorylated epitopes of neurofilaments have been conserved during chordate evolution

We have characterized some rabbit polyclonal responses as strictly specific for phosphorylated epitopes located in the carboxyterminal (tail) domain of the H or the M subunits of mammalian neurofilaments. These antibodies have been used to confirm the occurrence in lizard neurofilaments of a single heavy subunit cross-reacting with both H and M from mammals. A heavy subunit with similar cross-reactivity has been detected in neurofilaments preparations from fishes, whereas more primitive Chordata possess a HMW polypeptide cross-reacting with only the M subunit. We could also demonstrate in frog spinal cord two distinct heavy subunits cross-reacting with either the M or the H subunit from mammals, a fact which suggests a convergent evolution for phosphorylated epitopes of neurofilaments.     

Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures

We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.     

Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.     

Antibodies against neurofilament subunits label retinal ganglion cells but not displaced amacrine cells of hamsters.

Although neurofilament (NF) antibodies have been used to visualize ganglion cells and their axons in the retina, it is not known, however, how many ganglion cells contain NF, and how the various NF subunits are distributed in the ganglion cells. Moreover, it is not known whether displaced amacrine cells in the ganglion cell layer are also labelled. In order to see whether NF antibodies can be used as a specific marker for ganglion cells, antibodies raised against the low (NF-L), middle (NF-M) and high (NF-H) molecular weight subunits of NF were employed to stain retinal whole-mounts of adult hamsters after pre-labelling the ganglion cells with Granular Blue. It was found that NF-L and NF-H antibodies labelled 38,777 and 17,750 cells in the ganglion cell layer respectively. By co-localization with GB-labelled cells, 88% of NF-L positive cells and 91% of NF-H positive cells were found to be ganglion cells. In contrast, the NF-M antibody labelled only very few ganglion cells (418 per retina) although robust staining of axonal bundles was observed. Thus, NF antibodies may prove useful in studying this population of ganglion cells.     

Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons

Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of approximately 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in approximately 19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF- L subunits. Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

Two isozymes of the Na,K-ATPase have distinct antigenic determinants

Two isozymes of the Na,K-ATPase were purified from rat renal medulla and rat brainstem axolemma, and antisera were raised in rabbits. When antibody titers were measured, two sera showed specificity for either the kidney or axolemma Na,K-ATPases and had limited cross-reactivity which could be removed by cross-adsorption. In blots of polyacrylamide gels, these sera reacted with only the alpha or alpha (+) Na,K-ATPase catalytic subunits, while they cross-reacted with both types of beta subunits. Two other sera each recognized both alpha and alpha (+), indicating that the catalytic subunit isozymes have additional shared antigenic determinants. A comparison of the Na,K-ATPases from the brains of different vertebrate species indicates that birds and fish differ from mammals and amphibians in the manifestation of Na,K-ATPases isozymes. Neither neuraminidase nor endoglycosidase F treatment eliminated specific antibody reaction or affected the electrophoretic mobilities of the alpha and alpha (+) subunits, although endoglycosidase F increased the mobilities of the two types of beta subunits to similar final apparent molecular weights. Blots of the peptide fragments produced by incomplete papain and trypsin digests of the alpha and alpha (+) subunits were stained with the specific sera, and the patterns of immunoreactive fragments were found to be markedly different. The results suggest that the antigenic differences reside in differences in the primary protein sequences of the two isozymes.     

Acrylamide alters neurofilament protein gene expression in rat brain

Acrylamide, a prototype neurotoxin, alters neurofilament protein (NF) gene expression in rat brain. Levels of mRNA coding for neurofilament protein subunits NF-L, NF-M, and NF-H have been determined by Northern blot analysis using³²P-labeled cDNA probes. Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls. The expression of NF-L or NF-H mRNA is not affected by acrylamide. In contrast, chronic treatment with acrylamide [0.03% (w/v) in drinking water for 4 weeks] induces a modest but significant increase (approximately 22%) in NF-L mRNA compared to controls. Levels of NF-M, and NF-H mRNA are not altered by acrylamide treatment. The expression of -actin mRNA, an ubiquitous protein, is not affected by either treatment regimen of acrylamide. The results of this study show that acrylamide increases the expression of mRNA for NF protein subunits in rat brain. The increase of specific mRNA for NF subunits depends on the dose, duration and route of acrylamide administration.     

Requirement of Heavy Neurofilament Subunit in the Development of Axons with Large Calibers

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.     

Binding of Bodian''s Silver and Monoclonal Antibodies to Defined Regions of Human Neurofilament Subunits: Bodian''s Silver Reacts with a Highly Charged Unique Domain of Neurofilaments

Cleavage at cysteine and chymotrypsin digestion were applied to two human neurofilament (NF) subunits, low- and high-molecular-weight NF (NF-L and NF-H), to locate the regions reacting with Bodian's silver stain and with several monoclonal antibodies, including NF-specific antibodies and one that recognizes all intermediate filaments (anti-IFA). Our findings indicate that whereas anti-IFA recognizes the highly conserved rod domain, all the NF-specific antibodies, as well as Bodian's silver, react with the carboxy-terminal tailpiece of NF subunits. The silver binding sites in NF-L are located in a carboxy-terminal 12-Kd chymotrypsin fragment, a highly charged, unique domain of NF.     

Distribution of neurofilament subunits in neurons and neuronal processes: immunohistochemical studies of bovine cerebellum with subunit-specific monoclonal antibodies

The distribution of individual neurofilament (NF) subunits in bovine cerebellar neurons was examined using monoclonal antibodies (MAs) raised against bovine NF. MAs with immunochemically defined specificities for one or more NF subunits were used. Seven were specific for the Mr 68,000 NF subunit, five were specific for the Mr 150,000 NF subunit, nine were specific for the Mr 200,000 NF subunit, and 30 recognized both high molecular weight subunits. Fresh bovine cerebellum was fixed and processed by five different protocols and subjected to four different immunohistochemical procedures. MAs from each group stained neuronal perikarya and processes. NF immunoreactivity in Purkinje cells was evaluated in detail. Adjacent Purkinje cell bodies and dendrites exhibited variable NF immunoreactivity to the same MA, ranging from intensely positive to completely negative. Similar variability in axonal staining was not observed. Application of the same MA to tissue subjected to different fixation and/or immunohistochemical protocols also resulted in variability in NF subunit immunoreactivity. We conclude that MAs recognize each of the three NF subunits in neuronal perikarya, axons, and dendrites. Variability in NF subunit immunoreactivity appears to reflect both NF microheterogeneity and fixation-dependent modifications of NF subunits.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

Effect of conformation of hCG-beta on generation of hCG-specific antibody

Most antisera generated to isolated highly purified beta subunits of human glycoprotein hormones are not sufficiently sensitive to detect physiologic blood levels of the native hormone. In the dissociated state, beta subunits assume a conformation different from that in the native hormone. Since antisera to alpha subunits have essentially no cross-reactivity between species, highly purified hCG-beta was combined with bTSH-alpha. That hybrid served as immunogen to assess whether sensitive, specific hCG antisera would more likely result than using hCG-beta alone. Of five animals immunized, three developed sufficiently sensitive and specific antisera. The results of these studies strongly suggests that human glycoprotein beta subunits combined with non-human alpha subunit are more likely to yield specific, sensitive antisera than when either isolated beta subunit or the native human glycoprotein hormone, containing common alpha determinants, serves as immunogen.     

Identification of a neurofilament-like protein in the protocerebral tract of the crab <Emphasis Type="Italic">Ucides cordatus</Emphasis>

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of 160 kDa, similar to the mammaliam NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.This work was supported by CNPq, FAPERJ, CAPES and FUJB/UFRJ.     

Two separate receptors for prolactin in the rabbit mammary gland

Rabbit mammary gland PRL receptors in the microsome fraction were solubilized with the zwitterionic detergent Chaps, and were separated into two fractions (Fr. A and B) by ion-exchange chromatography. The number of receptors in Fr. B was about 2.2 times greater than in Fr. A. In sucrose gradient centrifugation analysis, PRL receptors in Fr. A and Fr. B sedimented at different positions. After binding 125I-PRL, the apparent molecular weight (mol wt) of the PRL receptor in Fr. A changed from 42,400 to 65,500 and that in Fr. B changed from 89,400 to 108,000, suggesting that each binding subunit interacts with one PRL molecule. Cross-linking 125I-PRL to receptors revealed little change following SDS-PAGE, in the autoradiogram patterns of the microsome PRL receptors, either in the presence or absence of dithiothreitol. Both the microsome and the Chaps extract contained two major binding subunits (mol wt, 83,200 and 36,800) and one minor subunit (mol wt, 20,800). The mol wt of the dominant PRL receptors in Fr. A and Fr. B were 36,800 and 83,200, respectively. The latter form did not dissociate into a 36,800 mol wt form, suggesting that the rabbit mammary gland contains two independent binding subunits with mol wt of 36,800 and 83,200. Data showed that PRL receptors in the rabbit mammary gland are mostly the high Kd type receptor with a mol wt of 83,200.     

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.     

Contractile Proteins of a Benthic Fish. III. Subunit Composition of Myosin

SYNOPSIS. Ultracentrifugal and electrophoretic experiments arereported on the subunit composition of myosin from skeletalmuscle of a benthic fish, Coryphaenoides species. Coryphaenoidesmyosin undergoes extensive association in concentrated KGI solutionsat neutral pH, but sedimentation equilibrium experiments indicatethe presence of a small fraction (3%) of monomeric myosin withmolecular weight approximately 440,000. At pH 11, some of theaggregated myosin is dissociated, and monomeric myosin is itselfdissociated into a heavy component (410,000 mol wt) and a lightcomponent (14,000 mol wt) that comprises 5–7% of the protein.The lialkali component of Coryphaenoides myosin yields a singlepredominant band on cellulose acetate electrophoresis and SDS-ureaelectrophoresis in 9% acrylamide gel. The stoichiometric evidenceindicates that Coryphaenoides myosin contains two heavy chains(205,000 mol wt) and two light chains (14,000 mol wt) that areequivalent with respect to net electrostatic charge and molecularweight. Preparations of myosin obtained by direct extractionfrom muscle mince and by dissociation of actomyosin extractedfrom muscle mince also contain 5% of a 47,000 mol wt componentpresumably actin), traces of 34–36,000 mol wt component,and about 5.7% of low molecular weight material (10,000–15,000)that probably represents contaminant protein, although the possibilityof denatured nivosin subunits cannot be excluded.