Purification of insect myosin and α-actinin

Hammond, K S; Goll, Darrel E.

doi:10.1042/bj1510189

Cited by 20 publications

(6 citation statements)

References 22 publications

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“…Not only does the polypeptide have the same mobility as vertebrate 0~-actinin on SDS gels electrophoresed in the WeberOsborne (1969) buffer system, but it is recognized by polyclonal antibodies raised against vertebrate smooth muscle o~-actinin. Further, the protein has an amino composition closely similar to that of Lethocerus 0~-actinin (Hammond & Goll, 1975;Ericson, 1987). In indirect immunofluorescence microscopy studies, honeybee myofibrils treated with mAb o~ show intense, specific fluorescence at the Zband (Fig.…”

Section: Immunoblot Analysis Of Monoclonal Antibodiesmentioning

confidence: 93%

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Z-band proteins in the flight muscle and leg muscle of the honeybee

Saide

Chin-Bow

Hogan-Sheldon

et al. 1990

J Muscle Res Cell Motil

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Monoclonal antibodies (mAb's) have been raised against proteins in preparations of Z-discs isolated from honeybee fibrillar flight muscle. These antibodies have identified four Z-disc antigens on immunoblots of honeybee fibrillar proteins. Antibody alpha binds to the 90-100 kD protein, alpha-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (greater than 600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAb's recognize previously uncharacterized insect Z-band proteins. Monoclonal antibody Z(400) binds to a pair of proteins with molecular masses near 400 kD and 600 kD. Antibody Z(175) recognizes two components, 158 kD and 175 kD, that are not only immunologically similar but have nearly identical peptide maps. Indirect immunofluorescence microscopy studies show that the proteins recognized by mAb's alpha, Z(175) and Z(400) are located at the Z-band, while the mAb P antigen is found on either side of it. Three of the four antibodies we have obtained recognize leg muscle proteins. Monoclonal antibodies alpha and P comigrate on SDS gels with analogous components from flight muscle. Only the smaller of the two proteins identified in flight muscle by mAb Z(400) is found in leg muscle, however. Furthermore, no Z(175) antigens have been detected in the non-fibrillar tissue by either monoclonal or polyclonal antibodies. Immunofluorescence microscopy studies localize the alpha and Z(400) antigens at the Z-line in leg muscle fibrils. Surprisingly, however, mAb P binds within the A-bands of synchronous fibres, not between the A- and Z-bands as in asynchronous fibrillar muscle.

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Section: Immunoblot Analysis Of Monoclonal Antibodiesmentioning

confidence: 93%

“…proteins have been identified (Saide & Ullrick, 1974;Bullard & Sainsbury, 1977;Hammond & Goll, 1975;Sainsbury & Bullard, 1980); their mode of assembly has not been determined. Moreoever, it is not known what factors dictate the Z-band structure found in a given tissue.…”

Section: Introductionmentioning

confidence: 97%

Z-band proteins in the flight muscle and leg muscle of the honeybee

Saide

Chin-Bow

Hogan-Sheldon

et al. 1990

J Muscle Res Cell Motil

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“…Protein binding assays Lethocerus actin and myosin were prepared as previously described (Hammond and Goll, 1975;Bullard et al, 1985); the actin preparation contained arthrin (a ubiquitinated actin; Ball et al, 1987) as well as actin. a-Actinin was prepared from rabbit skeletal muscle (Arakawa et al, 1970).…”

Section: Northern Blot Analysismentioning

confidence: 99%

Kettin, a large modular protein in the Z-disc of insect muscles.

et al. 1993

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Z‐discs of insect flight muscle contain a large protein of 500–700 kDa. Monoclonal antibodies label an epitope in the molecule at the Z‐disc in Drosophila and Lethocerus (waterbug). A partial cDNA of 1.6 kb from the Drosophila gene has been cloned and sequenced. The corresponding amino acid sequence has a modular structure composed of four conserved repeats of 95 amino acids homologous to immunoglobulin C2 domains (called class II domains in muscle proteins), separated by less conserved linker sequences of 35 amino acids. An expressed class II domain with flanking linker sequences binds to actin and alpha‐actinin but not to myosin. Single molecules of the protein would be large enough to span the Z‐disc. We suggest that the protein acts as scaffolding in the Z‐disc and we call the protein kettin. The Ca2+ activated protease, calpain, disrupts the Z‐disc of striated muscle, releasing alpha‐actinin intact. Calpain digests kettin to a series of peptides of between 30 and 170 kDa which are released from the myofibril. Digestion of kettin may cause disintegration of the Z‐disc and alpha‐actinin release which lead to disassembly of the myofibril.

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“…expressed as residues/ 1000, and the number of observations is 6. The values in column (B) are mean amino acid contents for the following myofibrillar proteins: Lethocerus myosin (Hammond & Goll, 1975); dung-beetle paramyosin (Bullard et al, 1973b); Lethocerus a-actinin (Hammond & Goll, 1975;Lethocerus actin (Bell, 1979); dung-beetle tropomyosin (Bullard etal., 1973b). residues/1000 in isolated honey-bee Z-discs (Saide & Ullrick, 1974).…”

Section: Results and Discussion Isolated Z-discpreparationmentioning

confidence: 99%

New proline-rich proteins in isolated insect Z-discs

Sainsbury¹,

Bullard²

1980

Biochemical Journal

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Z-discs were isolated from Lethocerus (waterbug) flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. Sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis confirmed a previous report that major Z-disc proteins had subunit mol.wts of 200 000, 180 000, 105 000, 95 000, 42 000 and 35 000. A protein of subunit mol.wt 25 000 was found in once-washed Z-discs but was degraded or was removed by successive washes. In addition, a protein of high molecular weight (less than 300 000) was found in Z-discs. Proteins of subunit mol.wts. 42 000, 35 000 and 25 000 were individually sliced from SDS/polyacrylamide gels and eluted. Amino acid analysis showed that the 35 000-subunit-mol.wt. protein was not, as was previously suggested, tropomyosin, but was a distinct Z-disc protein rich in proline. Calculations based on the amino acid analysis showed that this protein contained substantial hydrophobic regions. Preliminary investigations into the isoelectric point and a method of isolation of the 35 000-subunit-mol.wt. Z-disc protein are described. This protein was found in slices cut from SDS/polyacrylamide-gel electrophoretograms of whole myofibrils. The protein of 42 000 subunit mol.wt. was shown by amino acid analysis to be actin and the 25 000-subunit-mol.wt. Z-disc protein was proline-rich.

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Purification of insect myosin and α-actinin

Abstract: A method is described for preparing insect myosin, tropomyosin and alpha-actinin. The amino acid compositions of the myosin and alpha-actinin are given, and some of the properties of the purified proteins are discussed.

Cited by 20 publications

References 22 publications

Z-band proteins in the flight muscle and leg muscle of the honeybee

Z-band proteins in the flight muscle and leg muscle of the honeybee

Kettin, a large modular protein in the Z-disc of insect muscles.

New proline-rich proteins in isolated insect Z-discs

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