20K Fragment Research Articles

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture.

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1.

Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins.

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg2+-ATPase activity and the initial Pi burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.

Solubilization and Degradation of Extracellular Matrix by Various Metastatic Cell Lines Derived From a Rat Rhabdomyosarcoma<xref ref-type="fn" rid="FN1">2</xref>

Examination by use of WAG syngeneic female rats was made on 4 rat rhabdomyosarcoma sublines expressing different metastatic potentials for their abilities to degrade proteoglycans and glycoproteins of the extracellular matrix (ECM), deposited by corneal endothelial cells and metabolically labeled with [3H]glycosamine and [35S]sulfate. Of the label incorporated in ECM, 10-20% was released in the culture medium in 3 days by the cell lines studied. The proteoglycans, glycosaminoglycans, and glycoproteins released by the cells from ECM were separated and partially characterized. Most of the ECM glycoproteins were recovered in the region of 200,000 (200K) on gel chromatography. Glycopeptides of 10K-20K apparent molecular weight were minor components. The glycosaminoglycan chains of the proteoheparan sulfate were partially hydrolyzed to 20K fragments. Tumor cells increased also the solubilization of the nondegraded matrix macromolecules. About 5-8% of the radioactivity of the ECM remained associated with the tumor cells detached from the ECM. ECM degrading activity of the sublines did not correlate with their ability to colonize the lungs after iv injection but did correlate with their ability to metastasize to the lungs from the primary subcutaneous site.

Cross-linking of actin to myosin subfragment 1 in the presence of nucleotides.

Chemical cross-linking of actin to the 20K and 50K fragments of tryptically cleaved myosin subfragment 1 (S-1) by the zero-length cross-linking reagent 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC) was used as a probe of the acto-S-1 interface in the presence of nucleotides. The course of the two reactions was monitored by measuring on sodium dodecyl sulfate (SDS)-polyacrylamide gels the time-dependent formation of the 20K-actin and 50K-actin cross-linked products. Both reactions were inhibited somewhat in the presence of MgADP, were slowed 3-4-fold in the presence of magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and proceeded at least 7-fold slower with N,N'-p-phenylenedimaleimide (pPDM) modified S-1, as compared to the respective rates in the absence of nucleotides. However, neither the binding of the nucleotides MgADP and MgAMPPNP to S-1 nor the modification of S-1 by pPDM significantly changed the ratio of the cross-linking rates of actin to the 20K and 50K fragments. Similar to what was previously observed in the absence of nucleotides [Chen, T., Applegate, D., & Reisler, E. (1985) Biochemistry 24, 137-144], actin was cross-linked at an approximately 3-fold faster rate to the 20K fragment than to the 50K fragment under all reaction conditions tested. Thus, irrespective of the extent of acto-S-1 dissociation or the binding of nucleotides to acto-S-1, the 20K fragment remains the preferred cross-linking site for actin. These results show that the interaction of actin with each of the cross-linking sites on S-1 is not under selective or preferential control by nucleotides.

Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin.

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.

Purification and characterization of the respiratory syncytial virus fusion protein.

The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products.

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.

Photosensitized direct cross-linking of fluorescent analogs of ATP to the adenine recognition domain in myosin ATPase.

To obtain information about the adenine recognition site in myosin ATPase, ribosemodified fluorescent analogs of ATP, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives, were directly cross-linked to myosin subfragment-1 (S-1) ATPase by irradiation with visible light in the presence of FMN as a photosensitizer. The cross-linking of the fluorescent nucleotides was inhibited by addition of ATP or ADP. Tryptic digestion of the cross-linked S-1 revealed that fluorescence of the analog was associated predominantly with the 50K fragment and its precursor, the 75K one, and slightly with the 20K fragment. However, fluorescence was scarcely associated with the 26K fragment. The results were confirmed by cross-linking experiments using trypsin-split S-1, which mainly consists of the 50K, 26K, and 20K fragments. These findings suggest that the adenine recognition site of the myosin ATPase is located predominantly on the 50K domain.

Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1

The K +-EDTA-activated ATPase activity of chymotryptic myosin subfragment-1 (S-1) decreased by 85–90% when S-1 was incubated over a 2-h period at 35 °C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PP i, to S-1 before incubation at 35 °C prevented the loss of ATPase activity. The decrease in ATPase activity was also accompanied by changes in tryptic sensitivity. Instead of the normal peptide pattern—which is comprised of three heavy chain fragments (27K, 50K, and 20K)—only two fragments (27K and 20K) appeared on the sodium dodecyl sulfate-gel electrophoregram after limited tryptic digestion of thermally treated S-1. Addition of any ligand—e.g. ATP, ADP, pyrophosphate, or actin—which prevented the loss of ATPase activity during incubation at 35 °C also prevented the observed change in the tryptic peptide pattern of S-1. Tryptic digested S-1, whose heavy chain has been cleaved to 27K, 50K, and 20K fragments, also lost its ATPase activity upon mild heat treatment. The heat-treated trypsin-digested S-1 was subjected to a second tryptic digestion, which resulted in the disappearance of the 50K fragment, while the 50K fragment of tryptic S-1 not subjected to heat treatment was not susceptible to additional tryptic hydrolysis. The results indicate that the structural changes, that take place specifically in the 50K region of S-1 upon mild heat treatment, lead to both the loss of the ATPase activity and the changed tryptic sensitivity of S-1.

Direct cross-linking of three domains in the myosin head.

To obtain information about domain-domain contacts in the myosin head, trypsin-split myosin subfragment-1 (S-1), which mainly consists of 50K, 26K, and 20K fragments, was cross-linked directly by the photodynamic technique under mild conditions (pH 7-8.5, 0 degrees C) using riboflavin 5'-phosphate, protoporphyrin, and methylene blue as sensitizers. Exposure of trypsin split-chymotryptic S-1 to visible light in the presence of the sensitizers resulted in formation of cross-linked products of (20 + 26)K, (20 + 50)K, (26 + 50)K, and (20 + 26 + 50)K fragments. The results suggest that three domains in the myosin head are in contact with each other, at least partly, raising the possibility that the communication between the ATP- and actin-binding sites passes through a "short cut," i.e., the contact region between the domains.

Interaction of hemoglobin with band 3: A review

The oxygen transport protein hemoglobin interacts specifically and reversibly with the red cell membrane. pH and ionic strength dependence of these interactions indicate their electrostatic nature. The anion transport protein band 3 has been implicated as the protein to which hemoglobin binds. Hemoglobin, aldolase and glyceraldehyde 3-phosphate dehydrogenase have a similar pH and ionic strength dependence in binding to 23K fragment. The three compete for the same amino-terminal 23 residue sequence region of band 3. The binding site is a highly acidic segment without any positive charge. We have recently determined the sequence of amino-terminal 23K fragment of band 3. There is a remarkable internal sequence homology between the first eleven and next eleven residues in this sequence region. Biophysical measurements in this sequence region. Biophysical measurements have revealed that 23K is a tetramer under physiological conditions. The implications of this structure of 23K is discussed with respect to the interaction of band 3 with the red cell cytoskeleton.

An actin-binding site on the 20K fragment of myosin subfragment 1

Myosin subfragment 1 (S1) was covalently labeled with a fluorescent dye, N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM), and then digested by trypsin to cleave S1 heavy chain into fragments. The DACM-labeled and trypsin-treated S1 was complexed with F-actin and treated with a zero-length cross-linker, 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide (EDC). The cross-linking reaction generated a covalently linked complex of actin and the 20K fragment of S1 heavy chain, which exclusively incorporated the fluorescent dye, to form a fluorescent 65K cross-linked product. The 20K and 65K fluorescent peptides were isolated and purified and then subjected to cyanogen bromide and/or hydroxylamine cleavages. Mapping of fluorescent cleavage products on acrylamide gels revealed that the N-terminal 20 residues of the 20K fragment of S1 heavy chain contained a cross-linking site of actin.

Reactive lysyl of myosin subfragment 1: location on the 27K fragment and labeling properties.

The limited tryptic digestion of the heavy chain of chymotryptic myosin subfragment 1 resulted in five peptides with approximate molecular weights of 75K, 50K, 29.5K, 27K, and 20K. Of the five peptides, two, 75K and 29.5K, were transient and disappeared during the digestion. Our data suggest that the 27K fragment is generated by two parallel routes: directly from the 75K fragment and through a 29.5K precursor. A method was developed to isolate the final products, 50K, 27K, and 20K fragments, of the tryptic hydrolysis of the heavy chain of myosin subfragment 1. Using this method, it was found that the reactive lysyl residue, labeled by a trinitrophenyl moiety, resides in the 27K fragment. The reactive lysyl residue was also present in the 29.5K fragment. The trinitrophenylation of the reactive lysyl residue was inhibited by magnesium pyrophosphate in the 27K but not in the 29.5K fragment. This may indicate that the two routes of generating the 27K peptide correspond to the proteolysis of two qualitatively different subfragment-1 heads as suggested by Tonomura [Tonomura Y. (1972) Muscle Proteins, Muscle Contraction and Cation Transport, University of Tokyo Press, Tokyo, and University Park Press, Baltimore].

Location of SH1 and SH2 along a heavy chain of myosin subfragment 1.

Two reactive SH groups (SH1 and SH2) of myosin subfragment 1 (S-1) were selectively labeled with a fluorescent dye, N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM). When the DACM-S-1 was digested with trypsin, 95K heavy chain was cleaved into 50K, 25K, and 20K fragments, and the fluorescent labels incorporated into SH1 and SH2 were exclusively found in the 20K fragment, indicating that these SH groups were located in the fragment. When the trypsin-treated DACM-S-1 was subsequently fragmented with hydroxylamine in 6 M guanidine hydrochloride at pH 9.0, the fluorescent 20K fragment was cleaved into two fluorescent segments, the 13K segment containing SH1 and the 5K segment containing SH2. Since it is known that SH1 and SH2 are only 10 residues apart in the sequence and that SH1 is nearer the COOH terminus than SH2, the results show that these reactive SH groups are separated from the COOH terminus of S-1 heavy chain by a 13K-dalton stretch of polypeptide chain.

Structural aspects of actomyosin interaction

Actin binding to myosin-S1 modulates the limited tryptic cleavage of the COOH-terminal region of the 95K heavy chain at the joint connecting the 75K and 20K peptide units; concomitantly actin affords total protection against the resulting loss of acto-S1 Mg2+-ATPase activity. The specificity of the actin effect is illustrated by the fact that it exerts itself not only on free S1 but also on the intact myosin molecule. Mg2+-ATP and Mg2+-ADP impair the protective action of actin to an extent closely related to their respective affinity for the acto-S1 complex. Tryptic fragmentation of S1 heavy chain under highly controlled conditions, using trypsin to S1 weight ratios in the range 1:1000 - 1:1500 led us to establish that peptide bond cleavage at the 75K-20K junction is a sequential process giving rise first to a 22K peptide intermediate which is subsequently converted to the stable 20K fragment. Most importantly, it is also demonstrated that the loss of S1 activation by actin is not due to the initial scission of the 75K-22K linkage but is intimately associated with the breakdown of the 22K precursor into its 20K moiety. Three trypsin-modified S1 derivatives, the heavy chain of which is a complex of two or three fragments, were purified. A detailed analysis of the C-termini of these fragments, as compared to the C-terminal structure of the intact heavy chain, indicated that the 20K fragment is formed mainly through the degradation of a NH2-terminal 2K segment in the 22K precursor and that this proteolytic event is the only one accounting for the acto-S1 ATPase loss. Cross-linking experiments exploiting the reaction of a carbodiimide reagent with rigor complexes containing either fluorescent actin or fluorescent fragmented S1 revealed unequivocally the attachment of the actin monomer to recognition sites on the 20K and 50K units of S1 heavy chain. Specific interactions between the C-terminal 20K domain and light chain LC2 are proposed as being part of the molecular mechanism of the myosin-linked regulation of actomyosin interaction.

Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase.

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.