Biomedical subjects
Publications and source records attributed to J Trinick.
Titin and nebulin are giant muscle proteins, both of which are approximately 1 micron long and are composed of many repeating domains. Titin domains resemble type III fibronectin and C-2 immunoglobulins. Both proteins are likely to be involved in specifying and stabilizing the highly ordered structure of muscle, probably by acting as 'protein rulers' to regulate the assembly of myosin and actin filaments precisely.
The structure of the complex of actin and myosin subfragment-1 (S1) during steady-state ATP hydrolysis has been examined by electron microscopy. This complex is normally dissociated by ATP in vitro but was stabilized here by low ionic strength. Optimal conditions for attachment were established by light-scattering experiments that showed that approximately 70% of S1 could be bound in the presence of ATP. Micrographs of the unstained complex in vitreous water suggest that S1 attaches to actin in a variety of configurations in ATP; this contrasts with the single attached configuration seen in the presence of ADP. The data are therefore compatible with the idea that a change in attached configuration of the myosin cross-bridge is the origin of muscle force. In control experiments where ATP was allowed to hydrolyze completely the binding of the S1 seemed cooperative.
Molecules of the giant protein titin are responsible for the passive elasticity and central A-band location of muscle myofibrils. The molecular mechanism of titin elasticity is not known, but the I-band region of the molecule appears capable of approximately fourfold reversible extension. Such large extensions are likely to involve unfolding of titin domains. In the present experiments, equilibrium unfolding of titin from rabbit skeletal muscles was studied in vitro by fluorescence and circular dichroism spectroscopy, after addition of guanidinium chloride. The data suggest two unfolding transitions, both of which appear cooperative. The second transition is likely to involve complete unfolding of the immunoglobulin- and fibronectin-like domains from which the molecules is composed. The free energy associated with this transition is comparable with the energy required to extend titin molecules to the maximum amount seen in situ.
A simple solid-phase binding assay was used to screen for interactions that the giant myofibrillar protein titin makes with other sarcomeric proteins. The titin used in the tests was purified by a modified procedure that results in isolation of approximately 20 mg relatively undegraded protein in < 24 h. In addition to the approximately 3 MDa polypeptide, bands at approximately 160 kDa and approximately 100 kDa were also consistently seen on gels. Binding of titin to myosin, C-protein, X-protein and AMP-deaminase was observed. The interaction with myosin appears to be with the light meromyosin part of the molecule.
Individual molecules of the giant muscle proteins titin and nebulin span large distances in the sarcomere. Approximately one-third of the titin molecule forms elastic filaments linking the ends of thick filaments to the Z-line. The remainder of the molecule is probably bound to the thick filament where it may regulate assembly of myosin and the other thick filament proteins. This region also contains a sequence similar to catalytic domains in protein kinases. Nebulin appears to be associated with thin filaments and may regulate actin assembly.
Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M- to Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A-band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M-line encode a kinase domain and are closely related to the C-terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A-band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin-C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super-repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C-protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A-band titin is likely to be involved in the ordered assembly of the vertebrate thick filament.
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Partial amino acid sequence was obtained from the massive myofibrillar protein nebulin. This consists of repeating motifs of about 35 residues and super-repeats of 7 x 35 = 245 residues. The repeat-motifs are likely to be largely alpha-helical and to interact with both actin and tropomyosin in thin filaments. Nebulin from different species was found to vary in size in proportion to filament length. The data are consistent with the proposal that nebulin acts as a protein-ruler to regulate precise thin filament assembly.
Striated muscle is now known to contain a third major class of filaments, additional to the thick and thin filaments. The presence of such extra filaments has seemed likely for many years, but details of their location, structure, and composition are only now becoming clear. They are composed of massively large proteins and, in contrast to thick and thin filaments, they are elastic.
It is demonstrated that solutes are likely to be significantly concentrated under conditions commonly used to prepare vitrified aqueous suspensions for transmission electron microscopy. Muscle thick filaments in such suspensions were largely dissolved, probably due to an increase in salt concentration caused by evaporation of water immediately prior to freezing. The extent of solubilization indicated that salts had been concentrated by at least 50%. Simple tests showed that, under the conditions used, the rate of reduction in the height of a thin water layer was approximately 1 micron/s. Apparatus is described which reduces evaporation, by clamping the hydrated grid in a filter paper sandwich until just before it enters the ethane coolant. High-speed cine photography showed that, using this device, exposure of the thinned specimen to the atmosphere was approximately 200-fold less. Frozen-hydrated thick filaments prepared using the device had the expected length of about 1.6 microns.
The heads of myosin molecules from the striated adductor muscle of scallop have been studied by electron microscopy after negative staining. In common with vertebrate skeletal muscle myosin visualized by this method, the scallop myosin heads were pear-shaped and often showed pronounced curvature. Staining suggestive of two or, more frequently, three domains could often be observed. Removal of regulatory light chains (R-LCs) resulted in a reduction in the length of the heads of about 2.6 nm, with no significant change in maximum width. In desensitized preparations a majority of heads displayed anticlockwise curvature, whereas intact heads were usually seen curved clockwise. Analysis of the head curvature in both intact and desensitized molecules was consistent with an ability of each head to rotate about its long axis. Desensitization resulted in an increased incidence of heads showing two domains. It seems likely that the reduction in length upon removal of the R-LC is due to the two small domains located in the neck region of the head collapsing into one.
The protein titin has been localized by electron microscopy of myofibrils labelled with monoclonal antibodies. The data are consistent with individual titin molecules extending from near the M-line to beyond the ends of thick filaments, a distance of approximately 1 micron. In the A-band, titin appears to be bound to thick filaments, probably to the outside of the filament shaft. Molecules of titin in this configuration provided an obvious mechanism by which the length of thick filaments could be regulated accurately.
The effect of insulin-dependent diabetes on the hepatic microsomal activation of chemical carcinogens to mutagenic intermediates in the Ames test was investigated in rats pretreated with streptozotocin. In order to discern between the effects of streptozotocin itself and that of the resulting diabetes, groups of streptozotocin-treated rats received either nicotinamide simultaneously with the diabetogenic agent to prevent the onset of diabetes or daily treatment with insulin in order to antagonise the effects of diabetes. The activation of two nitrosamines, nitrosopiperidine and nitrosopyrrolidine was markedly increased following treatment of the animals with streptozotocin, the effect being preventable by nicotinamide and effectively antagonised by insulin. A similar increase in mutagenic response was also seen when Glu-P-1, a carcinogen generated during the cooking of proteinaceous food, was employed as the mutagen. In contrast, the diabetic rats were less efficient than control animals in activating the aromatic amine 2-aminofluorene to mutagenic intermediates. Concomitant administration of nicotinamide with streptozotocin prevented the decrease in mutagenicity, and daily treatment of diabetic rats with insulin partially restored mutagenic response to control levels. Streptozotocin-induced diabetes had no effect on the mutagenicity of 4-aminobiphenyl and the two polycyclic aromatic hydrocarbons, benzo(a)pyrene and 3-methylcholanthrene. The present findings clearly illustrate that diabetes modulates the metabolic activation of carcinogenic chemicals, the effect being dependent on the nature of the carcinogen.
Of the three isomeric forms of aminobiphenyl, only 4-aminobiphenyl is an established carcinogen while the 2-isomer is considered as a non-carcinogen and 3-aminobiphenyl is at best described as a weak carcinogen. In the present studies we investigated the mutagenicity of these three compounds, their N-hydroxy derivatives and their nitrosoderivatives in the Ames test using the Salmonella typhimurium strains TA98 and TA100. The studies were performed both in the absence and presence of an activation system derived from the liver of rats pretreated with Aroclor 1254. Of the three isomers only 4-aminobiphenyl exhibited mutagenicity and only in the presence of an activation system. N-Hydroxy-4-aminobiphenyl was a potent direct mutagen in both bacterial strains, N-hydroxy-2-aminobiphenyl was mutagenic in only TA100 while N-hydroxy-3-aminobiphenyl displayed mutagenicity in neither strain. Both 2- and 3-nitrosobiphenyls were direct mutagens in strain TA100. These findings suggest that the weak carcinogenicity of 3-aminobiphenyl may be attributed to the lack of genotoxicity of its N-hydroxyderivative, whereas in the case of 2-aminobiphenyl it may be due to the inability of the hepatic preparations to catalyse its N-hydroxylation, which is in agreement with published in vivo metabolic studies. It is interesting that of the three isomers only 2-aminobiphenyl is non-planar, forming a dihedral angle of 40 degrees, and this may preclude it from acting as a substrate of the P-450I family of haemoproteins, which selectively catalyses the N-hydroxylation of many aromatic amines including 4-aminobiphenyl.
The ability of the aza-aromatic polycyclic aromatic hydrocarbons 10-azobenz(a)pyrene and benz(a)acridine to induce the rat hepatic microsomal mixed-function oxidases was compared to that of their non-heterocyclic analogues benz(a)pyrene and benz(a)anthracene respectively. All four hydrocarbons markedly increased the O-deethylations of ethoxyresorufin and ethoxycoumarin, the non-heterocyclic analogues being the more potent. A more modest increase was seen in the O-dealkylation of pentoxyresorufin. All four hydrocarbons induced proteins recognised by antibodies to cytochrome P-450IAI but no increase was seen when antibodies to cytochrome P-450IIB1 were employed. The metabolic activation of benz(a)pyrene and Glu-P-1 to mutagenic intermediates in the Ames test was enhanced by all pretreatments. It is concluded that the aza-aromatic polycyclic hydrocarbons, like their non-heterocyclic analogues, selectively induce the cytochrome P-450I family of proteins.
Electron microscopy of negatively stained vertebrate skeletal muscle myosin molecules has revealed substructure suggestive of globular domains in the head portions of the molecule. This head substructure has been examined after both low and high electron doe. The results suggest it is probably not an artefact of radiation damage. The most common appearance is of one or two stain-filled clefts which run roughly perpendicular to the long axis of the head, giving rise to the appearance of two or three domains in a line. A large domain is located at the end of the head, while two smaller domains are arranged between this and the head-tail junction. The size of the large distal domain (about 10 nm long and about 7 nm wide at its widest point) is similar in heads showing either two or three domains. Stable analogues of M.ATP and M.ADP.Pi, the predominant complexes present during hydrolysis of ATP by myosin, were prepared by crosslinking the two reactive SH groups (SH1 and SH2) in the myosin head heavy chain with N,N'-p-phenylenedimaleimide (pPDM) in the presence of ADP, and by forming a complex with vanadate ion and ADP. At this resolution (approximately 2 nm) the heads of these modified molecules did not appear markedly different from those of the untreated protein, although there was a small increase in the number of straight as opposed to curved heads after cross-linking with pPDM.
The effect of chemically-induced diabetes on the hepatic microsomal mixed-function oxidase system and the activation of chemical carcinogens was investigated in animals treated with streptozotocin (STZ). In order to distinguish between the effects of the diabetogenic chemical per se and that of the diabetic state, groups of STZ-treated animals received either nicotinamide simultaneously with STZ to prevent the onset of diabetes, or daily treatment with insulin in order to reverse the effects of diabetes. STZ-treated animals exhibited higher pentoxyresorufin O-dealkylase, ethoxy-resorufin O-deethylase, ethoxycoumarin O-deethylase, aniline p-hydroxylase and NADPH-cytochrome c reductase activities; similarly, increases were seen in cytochrome P-450 and b5 levels. All of these effects were prevented by nicotinamide and, at least partly, antagonised by insulin therapy. Treatment of animals with STZ markedly increased the activation, by liver microsomes in vitro, of Trp-P-1 and Trp-P-2 to mutagens, the effect being totally preventable by nicotinamide and successfully antagonised with insulin therapy. The diabetic animals were similarly more efficient in activating MeIQ but the effect was not preventable by nicotinamide or reversed by insulin. In contrast no changes were seen in the activation of IQ and only a modest increase in the case of MeIQx. It is concluded that diabetes may modulate the metabolic activation of some chemical carcinogens, presumably by changing the ratio of the various cytochrome P-450 isoenzymes.
F-protein, a consistent contaminant of myosin preparations, has been shown to be phosphofructokinase, the key regulatory enzyme of glycolysis. In homogenates of rigor muscle most of the phosphofructokinase sediments with the myofibrils, suggesting that in the living muscle cell phosphofructokinase is not in the soluble fraction as was formerly thought, but bound to the myofibrils. Fluorescent antibody to F-protein labels myofibrils in a zone in each half of the A-band. The increase in separation of the zones across the A-band with increase in sarcomere length suggests that the antibody binds to the parts of the cross-bridge regions of the thick filament within the H-zone. It therefore seems likely that phosphofructokinase is located in the cross-bridge region of the thick filament, but that access of antibody is restricted by overlapping thin filaments.