Requirement of the 20-kDa light chain for the papain-resistant conformation of gizzard myosin

The limited chymotryptic digestion of unphosphorylated gizzard myosin in 0.15 M NaCl converted a papain-insensitive myosin in ATP to a papain-sensitive one. This conversion without phosphorylation of its 20-kDa light chain was accompanied with truncation of a 200-kDa heavy chain to a 195-kDa fragment and with the degradation of a 20-kDa light chain. Papain also yielded the 195-kDa fragment from the heavy chain, irrespective of the presence or absence of ATP. However, the ATP-induced protection of unphosphorylated myosin from the papain-digestion disappeared concurrently with degradation of the 20-kDa light chain by papain rather than the truncation of heavy chain. Papers from two laboratories [Onishi, H. & Watanabe, S. (1984) J. Biochem. (Tokyo) 95, 903-905; Kumon, A., Yasuda, S., Murakami, N., and Matsumura, S. (1984) Eur. J. Biochem. 140, 265-271] have reported that the ATP-protection of unphosphorylated myosin against papain is not observed after the 20-kDa light chain has been phosphorylated. The present results might indicate that the ATP-induced protection is also abolished through the chymotryptic degradation of the 20-kDa light chain.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

The calmodulin-binding domain on microtubule-associated protein 2

Microtubule-associated protein 2 (MAP2) binds calmodulin with a stoichiometry approaching 1-1.5 mol of calmodulin/mol of MAP2 in the presence of calcium ion. The calmodulin-binding domain(s) of MAP2 were probed by cross-linking 125I-calmodulin with partially digested MAP2, by limited digestion of the preformed 125I-calmodulin-MAP2 adduct, and by cross-linking 125I-calmodulin with the projection- and assembly-promoting portions of MAP2. Cross-linking 125I-calmodulin with partially digested MAP2 resulted in radioactive adducts of approximately 300, approximately 235, approximately 205, approximately 58, and approximately 40 kDa. The radioactive adducts with smaller molecular mass became prominent with increasing time of digestion concomitant with loss of those with higher molecular size. Limited chymotryptic digestion of preformed 125I-calmodulin-MAP2 adducts also produced a approximately 58-kDa radioactive band followed later by a approximately 40-kDa band. Brief chymotryptic digestion and subsequent centrifugation of microtubules preformed with pure tubulin and MAP2 permitted separation of microtubule-bound MAP2 fragments (molecular mass = approximately 215, approximately 180, and approximately 36 kDa) from unbound fragments (molecular mass = approximately 240, approximately 180, and approximately 140 kDa). 125I-Calmodulin cross-linked only with the microtubule-bound MAP2 fragments (forming mainly the approximately 58-kDa adduct) and not with unbound MAP2 fragments. Since the apparent molecular size of calmodulin is approximately 21 kDa on these sodium dodecyl sulfate-polyacrylamide gels, the results indicate that partial digestion of MAP2 by chymotrypsin produces a approximately 37-kDa fragment which can be further degraded to a approximately 20-kDa fragment. The approximately 37-kDa fragment that is labeled corresponds to the previously identified assembly-promoting fragment that attaches to the microtubule.     

Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site

Dictyostelium myosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V-8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V-8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with 32P was analyzed by SDS-PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp bends.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.     

Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain

The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 of the myosin heavy chain; among the residues of particular interest in this fragment are N epsilon-trimethyllysine, one of four methyl-amino acids in myosin, and Ser-324, which is photoaffinity labeled by an ATP analogue (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995). Combination of this sequence with those of the 23- and 20-kDa fragments yields an 809-residue sequence that constitutes most of the heavy chain of chymotryptic S-1 of this myosin.     

Dictyostelium myosin: characterization of chymotryptic fragments and localization of the heavy-chain phosphorylation site

Chymotrypsin cleaves Dictyostelium myosin in half, splitting the heavy chain (210,000 daltons) into two fragments of 105,000 daltons each. One of the two major fragments is soluble at low ionic strength and has a native molecular weight of 130,000. As judged by SDS polyacrylamide gel electrophoresis, this soluble fragment consists of the two intact myosin light chains of 18,000 and 16,000 daltons and a 105,000-dalton polypeptide derived from the myosin heavy chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin- activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin, although its apparent Km for actin is 12-fold greater than that of myosin. In addition to the major soluble 105,000-dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin also generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments that are devoid of myosin heads. A less prevalent insoluble fragment has a molecular weight of 83,000 and is probably a subfragment of the insoluble 105,000-dalton fragment. The heavy chain of myosin is phosphorylated in vivo and the phosphorylation site has been localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.     

Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.     

Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.     

Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin

Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin-activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non-phosphorylated form and single and double phosphorylated forms of this fragment were separated by two-dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non-phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.     

Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.     

Preparation of subfragment-1 from abalone smooth muscle myosin

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.     

Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids.

In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.     

Physical, enzymatic, and contractile properties of brain myosin with anti-brain myosin Fab fragment bound on its tail

An antibody obtained by immunizing a rabbit with purified bovine brain myosin was found to react with the tail portion of the myosin heavy chain. An Fab fragment obtained by limited papain digestion of the antibody was allowed to bind to brain myosin, and the complex of the Fab fragment and brain myosin (Fab-myosin) was isolated. On examination of the rotary-shadowed Fab-myosin by electron microscopy, most of the Fab fragment was located on the middle to C-terminal regions of the tails of the myosin molecules. The solubility of Fab-myosin in low salt solutions was higher than that of control brain myosin. Fab-myosin was found to form small irregular aggregates in low salt solutions instead of regular bipolar filaments, and the relative population of the monomeric form of myosin molecules observed for the Fab-myosin was much larger than that observed for the control myosin. The actin-activated Mg2+-ATPase activity of Fab-myosin was stimulated two- to threefold by phosphorylation of the light chains with myosin light chain kinase, as observed for the control brain myosin. Furthermore, the levels of the ATPase activity of the phosphorylated and dephosphorylated Fab-myosins were similar to those of the phosphorylated and dephosphorylated control myosins, respectively. The superprecipitation activity of Fab-myosin was also highly dependent on phosphorylation of the light chains. Although control brain myosin formed a large superprecipitate network which contracted to a dense particle, Fab-myosin generated only numerous tiny superprecipitates under the same conditions. From these results it was deduced that a regular filamentous state of brain myosin was not prerequisite for its actin-activated Mg2+-ATPase and superprecipitation activities but was indispensable for the formation of a large and well contractible superprecipitate.     

Functional domains of chicken gizzard myosin light chain kinase

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.     

Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate.

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.     

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.