Isolation, characterization and immunocytochemical localization of caldesmon-like protein from molluscan striated muscle – documents

Isolation, characterization and immunocytochemical localization

Aghlcb BARTEGI, Abdellatif FATTOUM, Caroline DAGORN, Jacqueline GABRION and Kidha KASSAB

Centre de Recherches de Biochimie Macromolbculaire du Centre National de la Recherche Scientifique,

Institut National de la Recherche Scientifique, Unite 249, Universitb Montpellier I, France

A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepiu officinalis was purified to

homogeneity and its main properties were investigated using biochemical and cytochemical approaches. Chicken masala recipe kerala style The

protein was found to be similar to chicken gizzard caldesmon. Chicken masala recipe italian It is a heat-stable protein.


Chicken masala recipe indian It cross-reacts immuno-

logically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2 +-dependent manner,

cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of

The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that

caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates.

However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under

our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar

bands or striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.

initiated by an increase in the intracellular free Ca2 ‘ concen-

troponin which binds to the actin-containing thin filaments in

vertebrate striated muscle [l]. Chicken marsala tyler florence (b) Calmodulin which reversibly

activates the myosin light chain kinase and thereby catalyses

the phosphorylation of the 20-kDa light chain of myosin. Chicken marsala crock pot recipe This

myosin-linked regulation is considered as the key event in the

muscle cells [2, 31. Chicken marsala casserole (c) The third system includes the 20-kDa

of regulatory mechanism. Chicken marsala sauce Recently, an actin-linked Ca2 ‘~ con-

arations of native thin filaments, and with synthetic thin fila-

present in substantial amounts in almost all types of vertebrate

Correspondence to A. Chicken marsala slow cooker recipe Fattoum, Centre de Recherches de Biochi-

biological activities similar to those known for the skeletal

zations of this protein extracted from scallop striated muscle.

cytochemically the caldesmon-like protein in various types of

volving the combination of several published procedures [17 –

30 pg/inl soybean trypsin inhibitor, 5 pg/ml leupeptin, 5 pg/

centrifuged at 12000 x g for 30 min at 4A°C. Chicken marsala recipe olive garden The clear super-

was then brought to pH 4.1 and left 30 min at 4A°C with gentle

stirring. Chicken marsala recipe It was sedimented at 3000 x g for 15 min at 4A°C to

[17]; gizzard caldesmon was further purified on a calmodulin-

Kiclley [20] and Weeds and Taylor [21], respectively. Chicken marsala emeril Protein

by Yin et al. Chicken marsala olive garden [25]. Chicken tikka masala in slow cooker One of the three tested polyclonal antibodies

striated inuscle were obtained as in [26]. Eye of the chicken Small pieces of tissue

fluorescence staining. Easy chicken tikka masala curry recipe They wcrc first incubated overnight in

goat anti-rabbit IgG (1 : 50 in phosphate buffered saline) pre-

epifluorescence microscope equipped with interferential filters

Fig. Indian chicken tikka masala curry recipe 1 . Chicken tikka masala recipe crock pot The distribution ojculdesmon-like components in the 30-60%

( N H 4 ) ZSO, fractions resulting .from heut-treated supernatants of

molluscan striuted muscle extracts. Chicken tikka masala calories restaurant Samples were subjected to SDS/

iminunovisualizcd (d -0. Chicken tikka masala calories Molecular inass markers (std): 200 kna

(myosin hcavy chain), 95 kDa (S-1 heavy chain), 67 kDa (serum albu-

min), 42 kDa (actin) and 14 kDa (lysozyme). Chicken tikka masala recipe indian Lane a: from the top to

the bottom of the gel, control purified filaniin, myosin hcavy chain,

caldesmon, cl-aclinin. Chicken marsala recipe with mushrooms actin and tropomyosin; l am b. Chicken marsala recipe rachael ray scallop muscle;

lanc c. Chicken marsala recipe martha stewart cuttle-fish musclc. Chicken marsala recipe carrabba’s Lanes d, e and f show the cross-reactivity

with the anti-(gizzard caldcsmon) antibodies of the samplcs in lanes

from heat-treated supernatants o f inuscle extracts and subject-

cuttle-fish (lanes c and f, striated muscles, of heat-stable pro-

Fig. Chicken marsala recipe food network 2. Chicken marsala recipe emeril lagasse Purification of caldesmon-like protein by ion-exchange chromatography. Chicken marsala recipe easy After treatment to remove tropomyosin (see Expcrimenlal

Procedures), the heat-stable scallop extract (150 mg protein) was chromalographed on DEAE-Sephacel (flow rate, 24 ml/h; fraction size,

3 ml). Chicken marsala recipe slow cooker The protein content of each [iaction was monilored at 280 nm and selected fractions (F, – F4) were examined every three tubes by

SDSPAGE (4-18% insct). Chicken marsala casserole paula deen The pool of F2 was submitted to a calmodulin-Sepharose column. Chicken marsala casserole rachael ray St, molecular mass markers. Chicken marsala casserole recipe myosin heavy

chain (200 kDa), S-I heavy chain (95 kDa), bovine serum albumin (67 kna), actin (42 kna), soybean trypsin inhibitor (21 kDa) and lysozyme

Fig. Chicken breast easy crock pot recipes 3. Chicken tikka masala in a slow cooker Calmodulin-Sepknrose uffinity chromatography of scallop caldesmon-like protein. Chicken marsala sauce in a jar (A) The FI fraction (30 mg protcin) from thc DEAE-

Sephacel column was dialyzed against 0.1 M NaCI, 1 mM MgCl2, 20 mM Tris/HCl, 0.1 mM dithiothreitol, 0.5 mM CaC12 (pH 7.5) and

loaded on a Ca2+-calmodulin-Sepharose column (1 x 10 cm) equilibrated with the same buffer (flow rate, 10 ml/h; fraction size, 1 ml). Chicken marsala sauce recipe The

absorbance profile of all fractions was monitored at 280 nm. Chicken marsala sauce without wine (B) 4- 18% gradient SDS polyacrylamide gel patterns of the whole F2 fraction

(a) and of the fraction retained by the column and eluted with 0.5 mM EGTA started at the point indicated by the arrow (b); (.c) EGTA-

eluted fraction immunostaincd with anti-(gizzard caldesmon) antibody

mon) polyclonal antibodies. Chicken marsala sauce packet The same cross-reactivity was caldesmon was visualized in the immunoblot (Fig. Chicken marsala sauce easy 1, lane d).

observed with the antiserum provided by Dr. Chicken marsala sauce with mushrooms Marston. Chicken marsala emeril lagasse We We conclude that the heat-stable material issued from the

tested the specificity of the anti-caldesmon antibodies using invertebratc muscles which cross-reacts with the anti-

filarnin, caldesmon, a-actinin, tropomyosin purificd from caldesmon antibody (Fig. Chicken marsala emeril recipe 1, lanes e, f , is an authentic

chicken gizzard smooth muscle and actin and myosin heavy caldesmon-related protein. Chicken marsala olive garden style The 240-kDa band, when it is pre-

chain from rabbit skeletal muscle (Fig. Chicken marsala olive garden calories 1, lane a). Chicken marsala olive garden recipe Only sent, could be an oxidized disulfide cross-linked form of

caldesmon which seems to have a higher proteolytic sensitivity

frozen or non-fresh muscle as a starting material, the protein

NaCl within the bulk of fractions 50 – 62, designated F2, well

within fractions F4. Chicken marsala olive garden nutrition The protein-staining patterns of electro-

Fig. Chicken marsala olive garden menu 2. Chicken tikka masala recipe easy Fraction F a , which is enriched in the 140-kDa protein,

Fig. Chicken garam masala recipe easy 5. Chicken tikka masala recipe with coconut milk Time-course of the change in the viscosity ofactin in thepresence

of scallop caldesmon-like protein. Stuffed chicken marsala recipe olive garden Polymerization of G-actin (1 4 pM)

was initiated by the addition of KCI to 100 mM in 20 mM TrislHC1,

(pH 7.0) and monitored by viscomctry at 20A°C. Best chicken marsala recipe olive garden ( A ) Polymerization

of actin alone; (A) polymerization of actin in the presence of scallop

caldesmon-like protein (molar ratio caldesmon/actin = 1 : 28)

Fig. Chicken calories and protein 6. Chicken tikka masala recipe indian style Inhibition of the Mg2+-ATPase activity cf skeletal actomyosin

hy the scallop caldesmon-like protein. How to make chicken tikka masala indian style Percentage inhibition or the

ATPase activity of rabbit skeletal muscle acto-S-1 in the absence (0)

tropomyosin/actin = 1 : 4) by increasing concentrations of scallop

a b c caldesmon-like protein (molar ratio, scallop caldesmon-like protein/

Fig. Chicken marsala recipe tyler florence 4. Food network chicken marsala tyler florence Binding of .~callop caldesmon-like protein to F-actin. What part of the chicken is gizzards The protein actin = 1 : 28; 1 : 14; 1 : 7 and 1 : 3). Healthy chicken easy recipes ( 0 ) Control acto-S-1 ATPase in

eluted from the Ca2 ‘-calmodulin -Sepharose column was the presence of gizzard tropomyosin and gizzard caldesmon. Chicken easy recipes for dinner The

cosedimcnted with F-actin (molar ratio, protein/actin = 1 :6) as indi- acto-S-I ATPase was also measured in the presence of scallop ( A ) or

cated in Experimental Procedures. Slow cooker chicken easy recipes The pellet (a) and supernatant (b) gizzard (V) caldesnion, 1 mM CaClz and calmodulin added at tenfold

were analyzed by gel elcctrophoresis; (c) gel profile ofthe supernatant molar excess relative to caldesmon. Ground chicken easy recipes 100% ATPase = 0.07 pmol Pi .

of the protein centrifuged in the absence of F-actin mg-‘ min-‘

Fig. Breast chicken easy recipes 7. Canned chicken easy recipes Cellular localization of caldesmon in d@rent types of muscles by immunofluorescence microscopy. Baked chicken easy recipes (A) Chicken gizzard smooth muscle;

(B) rabbit psoas, a fast muscle; (C) rabbit diaphragm, a mixed muscle; (D) rabbit semi-membranosus proprius, a slow muscle; (E) ventricular

cardiac muscle; (F) scallop striated muscle. Pictures of chicken gizzards By immunofluorescence, in longitudinal thin frozen sections of muscles, the protein was shown

in the sarcoplasm, in smooth muscle from chicken gizzard (A), with an intense and regular distribution. Chicken gizzard recipe In rabbit striated muscles (B-E),

the caldesmon was principally found in the sarcoplasmic elements (b), whereas no reaction seemed visible in the sarcomeric structure of the

myofibrils. Gizzard chicken The reaction was strengthened in the subsarcolemmal cytoplasm (+), around the nucleus (n), in these striated musclcs (see B and

C). Fried chicken gizzard recipe In the diaphragm (C) two kinds of distribution were observed in the sarcoplasm, allowing distinction of fast or slow (*) muscles cells, in

which caldesmon appeared localized as in B or D, respectively. Chicken gizzard nutrition The nucleus (n) was always negative. Chicken gizzard recipe chinese In scallop striated muscle (F), only the

tein, we investigated its interaction with rabbit skeletal actin.

interaction was measured by a cosedimentation-assay (Fig. Best chicken gizzard recipe 4).

terial bound to the affinity column cosedimented with F-actin.

with increasing concentration of the protein. Chicken gizzard soup The viscosity of

and cannot account for the observed viscosity increase. Chicken gizzard stew Scal-

described for gizzard caldesmon [31]. Chicken liver and gizzard recipe The overall results indi-

actomyosin in the presence of scallop or gizzard tropomyosin.

Fig. Gizzard of chicken 6 shows that scallop protein is able to inhibit the ATPase

activity of actomyosin. Gizzard chicken recipe Compared to the result obtained using

gizzard caldesmon as control, the extent of the inhibition was

roughly the same, as well as the release of this inhibition by

periodic fashion along stress fibers of cultured smooth muscle

this protein in striated muscle is not yet known. Chicken liver and gizzard To localize

sections were studied by indirect immunofluorescent labelling

using specific polyclonal antibodies elicited against chicken

cedures). Boiled chicken gizzard recipe The analysis of the cellular distribution confirms the

used as a control (Fig. Pickled chicken gizzard recipe 7A). Chicken gizzard recipes tenderizing However, in fast skeletal muscles,

the protein was less represented, and its distribution does not

seem to be related to the sarcomeric structures but rather to

the sarcoplasmic elements, particularly in the subsarcolemmal

interesting to note that caldesmon was distributed in the sarco-

plasm with different patterns according to the observation of

positive all over the cytoplasm and was as intense as in smooth

muscle sections. Chicken gizzard indian recipe The antibody to caldesmon interacted prefer-

entially with the antigen localized around the myofibrils like

localized at the periphery of the fibres (Fig. Spicy chicken gizzard recipe 7 F). Recipe for chicken liver and gizzard This specific

location of the protein, which is concentrated near the plasma

membrane in striated muscles, allows us to suggest its involve-

actin filaments to the cell membrane. Chicken gizzard salad Moreover, the identifi-

terial of about 45 kDa and could be oxidized and cross-linked

inhibition of actomyosin ATPase activity. Chicken gizzard soup recipe We have tentatively

byssus retractor muscle and in oyster muscles; they have pro-

the latch state observed in smooth muscles and these two tonic

‘This research was supported by grants from the C’enwe Nutiorid

de lu Recherche Scientijiyue, the Instirut Nurionul de lu Suntt. Gizzard of a chicken et de lu

Recherche MPdicak and the Association frunpzisr conire les Myopath-

caldesmon antibody and for many helpful discussions. Chicken gizzard and liver recipe We also thank

18. Easy chicken gizzard recipe Yaawa, M., Yagi, K . Gizzard in chicken & Sobuc, K. Chinese chicken gizzard recipe (1987) J . Chicken gizzard benefits Biochem. Chicken gizzard fried (Tokyc~)

19. What is a chicken gizzard Ngai, Ph. Quick chicken gizzard recipe K., Carruthers, C. Chicken gizzard curry A. Portuguese chicken gizzard recipe & Walsh, M. Chicken gizzard in chinese P. Chicken gizzard cooked (1984) Biochem.

20. Chicken liver gizzard recipe Eiscnberg, E. Best fried chicken gizzard recipe & Kielley, W. Simple chicken gizzard recipe W. Chicken gizzard gravy (1 974) J . Chicken gizzard protein Bid. Chicken gizzard chinese recipe Chem. Chicken gizzard cholesterol 249,4742-

21. Chicken gizzard price Weeds, A. Chicken gizzard plant G. Chicken gizzard stuffing & Taylor, R. Chicken gizzard fry indian recipe S. Chicken gizzard anatomy (1975) Nature (Lund.) 257. Chicken gizzard marinade 54-

irs. Chicken gizzard stir fry The authors wish to thank Dr. Chicken gizzard fry S. Chicken gizzard filipino recipe Marston for the gift of anti- 102, 1065-1073.

Dr. Healthy chicken gizzard recipe Fa??. Chicken gizzard recipe korean Bacou for expert advice on the different typcs of muscles. Chicken express fried chicken gizzard J. What part of the chicken is the gizzard 218,863 – 870.

Marston, S. Chicken gizzard stew recipe B. Baked chicken gizzard recipe &Smith, C. Chicken gizzard location W. Chicken gizzard adobo recipe J. Korean chicken gizzard (1984) J . Chicken gizzard recipes slow cooker Muscle Res. Chicken gizzard recipe south indian Cell Motil.

Marston, S. Chicken gizzard aralia B., Lehman, W.. Chicken gizzard calories Moody, C., Pritchard. Chicken gizzard recipe indian K. How to cook chicken liver and gizzard & Smith,

Ch.. Grilled chicken gizzard recipe Gilles, R. Puerto rican chicken gizzard recipe & Bollis, L., (eds) pp. Chicken gizzard recipe panlasang pinoy 69 -81, Springer-Verlag,

Sobue, K., Muramoto, Y., Fujita, M. Korean chicken gizzard recipe & Kakiuchi, S. Deep fried chicken gizzard recipe (1981) Proc.

Kakiuchi, S. Chicken gizzard nutrition facts & Sobuc, K. Is chicken gizzard healthy (1983) Trmds Bioclwm. What is a gizzard on a chicken Sci. Chicken gizzard slow cooker recipe 8, 9 – 6 2 .

Smith, C. What is a gizzard in a chicken W. Whats a chicken gizzard J., Pritchard, K. Chicken gizzard korean & Marston, S. Chicken gizzard salad recipe B. Chicken gizzard sisig recipe (1987) J . Chicken gizzard recipe kerala style Biol.

Ojima, T. What part of the chicken is a gizzard & Nishita, K. What is the gizzard of a chicken (1986) J . Chicken gizzard stew slow cooker Bioclirm. Is chicken gizzard healthy to eat (Tokyo) 100, 821 –

Fattoum, A., Bartegi. What part of a chicken is the gizzard A., Dagorne, C. Chicken gizzard gravy recipe & Kassab, R. Chicken gizzard recipes indian style (1989) J .

22. Chicken gizzard health benefits Lowry, 0. What’s a chicken gizzard H., Rosembrough, N. Chicken gizzard adobo J., Farr, A. What does a chicken gizzard do L. Gizzard chicken part & Randall, R. Aralia chicken gizzard J .

24. What is the chicken gizzard lowbin, H., Staehling. Function of gizzard in chicken T. Chicken gizzard function & Gordon, J. Chicken liver and gizzard adobo (1979) Proc. Chicken gizzard definition Nut1 Acud.

25. Chicken gizzard uk Yin, H. Where is the chicken gizzard located L., Albrecht, J. Chicken gizzard curry recipe H. Chicken liver and gizzard recipe indian & Fattoum, A. Where is the gizzard on a chicken (1981) J . Where is a chicken gizzard located Cell. What part of chicken is gizzard Biol.

28. What is a gizzard chicken Sobue, K., Muramoto. Chicken gizzard nutritional value Y., Fujita, M. Is chicken gizzard good for you &Kakiuchi. Chicken gizzard masala S. What part of chicken is the gizzard (3981) Pruc.

29. What part is the chicken gizzard Mornet, D.. Chicken gizzard pate Bertrand. What is gizzard of chicken R., Pantel, P., Audemard, E. Function of chicken gizzard & Kassab,

30. Chicken gizzard cooking time Lynch, W. Chicken gizzard images P.. What is the function of a chicken gizzard Riseman, V. Where is the gizzard located on a chicken M. How to clean a chicken gizzard & Bretscher, A. Gizzard function in chicken (1987) .I. What part of the chicken does the gizzard come from Biol.

31. What is the purpose of a chicken gizzard Galazkiewicz, B., Mossakowska, M., Osinska, H. What does a chicken gizzard look like & Dabrowska,

32. Adobo chicken liver and gizzard Furst, D. What part is a chicken gizzard O., Cross, R. Chicken gizzard in spanish A,, De Mey, J. Chicken gizzard nutritional info & Small, J. Chicken gizzard aralia plant V. What is a chicken gizzards function (1986)

33. What does the gizzard do in a chicken Bretschei-. Chicken gizzard dog treats A. Purpose of chicken gizzard & Lynch, W. Calories chicken gizzards (1985) J . Calories in chicken gizzards Cell Biol. Health benefits of eating chicken gizzard 100, 1656- 1663.

34. Recipe for pickled chicken gizzards Owada, M. Chicken gizzards rice and gravy K., Hakura, A., Lida, K., Yahara. Chicken gizzards pressure cooker I., Sobuc, K. Fried chicken gizzards nutrition &

Kakiuchi, S. Baked chicken gizzards and hearts recipes (1984) Proc. Fried chicken gizzards calories Natl Acad. Calories fried chicken gizzards Sci. Chicken gizzard plant care IJSA H I , 3133-3137.

35. Recipe for deep fried chicken gizzards Kakiuchi, R., Inui, M., Morimoto, K., Kauda, K., Sobue, K. Baked chicken gizzards &

36. Nutritional value of chicken gizzards Fujita, H., Ishimura, K., Ban, T., Kurosumi, M., Sobue, K. Chicken gizzards in pressure cooker &

37. How many calories in fried chicken gizzards Burgoyne, R. Calories in fried chicken gizzards D., Cheek. Calories in boiled chicken gizzards T. Chicken gizzards healthy R. Chicken gizzards and hearts &Norman, K. Best chicken gizzards M. Italian chicken gizzards (1986) Nature

sarcomeric muscles: basic and applied research prospects jbr the

90a??s (Carraro, U., ed.) pp. Health benefits of chicken gizzards 643-648, Unipress, Padova, Italy.

39. Benefits of chicken gizzards Marston, S. Recipe for fried chicken livers and gizzards (1989) J . Chicken gizzards for dogs Mu,scle Res. Oven baked chicken gizzards Cell. Chicken gizzards for catfish bait Motil. Best recipe for chicken gizzards 10, 97-100.

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