Isolation and characterization of casein kinases I and II from rabbit liver. Analysis of their effects on protein biosynthesis in the cell

Two cAMP-independent protein kinases isolated from rabbit liver extracts phosphorylate casein far more effectively than histones. The first protein kinase consists of one polypeptide chain (Mr = 37,000), utilizes exclusively ATP and is not inhibited in the presence of low heparin and RNA concentrations. The second protein kinase consists of three subunits (Mr = 42,000, 40,000 and 25,000 Da), utilizes both ATP and GTP and is inhibited by low heparin and RNA concentrations. The latter enzyme has Mr approximately 140,000 Da and possesses a polyanion-binding activity. These characteristics allow to relate the above enzymes to casein kinases I and II, respectively. Injection of casein kinase I into frog oocytes results in the inhibition of the rate of amino acid incorporations into the soluble and detergent extractable proteins. Casein kinase II has no effect on the amino acid incorporation into the recipient oocytes.     

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.     

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.     

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.     

The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.     

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.     

Separation and partial characterization of guinea-pig caseins.

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.     

Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification.

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polyacrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein bands (Mr 230000); secondly, two adjacent fast-moving protein band (M4 124000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230000 Mr as well as with that of 124000 Mr, but not with the polypeptide of 118000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80000-130000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mol per 237000 g protein. These results indicate that rat liver acetyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230000 and contains one molecular of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure.     

Immunochemical characterization of casein from rabbit mammary gland.

1. Excellent precipitating antibodies to rabbit recombined casein polypeptides were obtained in a sheep after 8 weeks of immunization with rabbit recombined polypeptides coupled to Sepharose-albumin. 2. The antiserum was assessed for specificity by several immunochemical techniques and was monospecific when tested against acid-precipitated casein, recombined casein and extracts of lactating rabbit mammary tissue. 3. A specific anti-casein immunoglobulin fraction was prepared by immunoadsorption of the antiserum by using Sepharose-recombined casein as immunoadsorbent. 4. The specific anti-casein immunoglobulin was used to prepare a Sepharose-anti-casein immunoadsorbent for the isolation of casein from extracts of rabbit mammary tissue.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum.

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences

Microtubules and microtubule-associated proteins (MAPs) have been isolated from cultured cells of Drosophila melanogaster by a taxol-dependent polymerization procedure. The principal MAPs are a group of four polypeptides with similar electrophoretic mobilities corresponding to approximately Mr 205,000 (the 205K MAP). These proteins are resistant to precipitation by boiling. One mouse monoclonal antibody and one polyclonal rabbit antiserum specific for the Mr 205,000 MAP were produced and characterized by immunoblotting and indirect immunofluorescence. Both antibody preparations stain the Mr 205,000 molecules and an Mr 255,000 molecule in immunoblots of Drosophila cell homogenates; the rabbit antiserum also stains an Mr 150,000 triplet. Both preparations stain the microtubules of the mitotic spindle, and the rabbit antiserum stains the cytoplasmic microtubules as well. Experiments using affinity-purified rabbit antiserum demonstrate that it is the Mr 205,000 species that is located in the mitotic apparatus and on cytoplasmic microtubules. A random shear genomic library was produced in the expressing vector lambda gt11 and screened with the rabbit antiserum to isolate the DNA sequences encoding these polypeptides. Several cross-hybridizing clones were recovered, shown to encode antigenic determinants in the Mr 205,000 MAP, and characterized by hybridization to Northern blots of mRNA and Southern blots of genomic DNA. Analysis by in situ hybridization reveals that the gene encoding the 205K MAP is located in polytene region 100EF.     

Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the beta chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of Mr 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the alpha chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by alpha and beta chain regions, respectively.     

Chemical structure and properties of duck and goose fibrinogen

Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented.     

Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.

RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.