Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Transmembrane topography of the mitochondrial phosphate carrier explored by peptide-specific antibodies and enzymatic digestion

Two peptides corresponding to the amino acid sequences 1-10 (N-terminal peptide) and 303-313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide, they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr approximately equal to 14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mr approximately equal to 17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter

Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-[3H]DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.     

Proteolytic and chemical dissection of the human erythrocyte glucose transporter.

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.     

Characterization of sulfated forms of the pro-opiomelanocortin amino-terminal glycopeptide in rat intermediate lobe cells

Explants of rat neurointermediate lobes were incubated in the presence of radioactive amino acids, sugars or sulfate and the labeled proteins were separated by two-dimensional gel electrophoresis. A double series of acidic peptides (Mr = 16,000-21,500) were identified as variant forms of the amino-terminal glycopeptide of pro-opiomelanocortin (N-POMC). The series of peptides with the higher molecular weights (Mr = 18,000-21,500) contain a tryptic fragment (tentatively identified as the tryptic peptide of the "joining peptide": sequence 77 to 93 of rat POMC) which is absent from the forms of the lower molecular weight series (Mr = 16,000 to 18,000). Pulse-chase studies further showed that the high molecular weight forms of N-POMC could be post-translationally cleaved albeit slowly into the species of Mr = 16,000-18,000 which constitute, at least in part, the final maturation products of the N-terminal region of the precursor molecule. All the variant forms of the N-POMC glycopeptide could be labeled with [35S]sulfate. Our results strongly suggest that most of the sulfate groups are attached to N-linked oligosaccharide side chains of N-POMC. We therefore propose that one of the final maturation products of the N-terminal portion of POMC in rat intermediate lobes is a sulfated glycopeptide (Mr = 16,000-18,000) composed of the 1-74 sequence of rat POMC.     

Site-specific antibodies as probes of the topology and function of the human erythrocyte glucose transporter.

Antibodies were raised against synthetic peptides corresponding to most of the regions of the human erythrocyte glucose transporter predicted to be extramembranous in the model of Mueckler, Caruso, Baldwin, Panico, Blench, Morris, Lienhard, Allard & Lodish [(1985) Science 229, 941-945]. Most of the antibodies (17 out of a total of 19) recognized the intact denatured protein on Western blots. However, only seven of the antibodies recognized the native membrane-bound protein, even after its deglycosylation. These antibodies, against peptides encompassing residues 217-272 and 450-492 in the hydrophilic central and C-terminal regions of the transporter, bound to the cytoplasmic surface of the erythrocyte membrane. This finding is in agreement with the prediction of the model that these regions of the sequence are cytoplasmic. Antibodies against peptides from the central cytoplasmic loop of the transporter were found to inhibit the binding of cytochalasin B to the membrane-bound protein, whereas antibodies against the C-terminal region had no effect. The anti-peptide antibodies were then used to map the sequence locations of fragments of the transporter arising from tryptic digestion of the membrane-bound protein. This in turn enabled the epitopes for a number of anti-transporter monoclonal antibodies to be located within either the central cytoplasmic loop or the C-terminal region of the protein. Of those monoclonal antibodies which inhibited cytochalasin B binding to the protein, all but one were found to have epitopes within the central region of the sequence. In conjunction with the results of the anti-peptide antibody studies, these findings indicate the importance of this part of the protein for transporter function.     

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.     

The glutamine residues reactive in transglutaminase-catalyzed cross-linking of involucrin

The protein involucrin, synthesized by human keratinocytes, contains 585 amino acids, largely in the form of 10 amino acid repeats, each containing glutamines in 3 conserved positions. Involucrin is a substrate for the keratinocyte transglutaminase and is labeled by the cosubstrate amine, glycine ethyl ester. Study of tryptic peptides of involucrin shows that a single glutamine (residue 496), located 89 residues from the C-terminal end, is preferentially labeled by the enzyme. Additional glutamine residues become reactive when the molecule is fragmented. The C-terminal end, isolated as a cyanogen bromide fragment of 275 residues, is labeled equally at 2 glutamine residues. The polypeptide containing residues 148 to 280 accepts practically no amine while in intact involucrin but as a free fragment is labeled at multiple glutamine residues. It is concluded that the C-terminal and N-terminal ends of the protein are directive influences in that they suppress the reactivity of a number of glutamine residues in the intact molecule, leaving one glutamine highly preferred by the transglutaminase.     

Biotin-conjugated reagents as site-specific probes of membrane protein structure: application to the study of the human erythrocyte hexose transporter

A novel labeling procedure using biotin-conjugated protein-modifying reagents has been employed to study the structure and function of the human erythrocyte hexose transporter. The carbohydrate moiety of the isolated, reconstituted transporter was labeled by using galactose oxidase/biotin hydrazide. Cysteine residues, which are essential for transporter function, were tagged with a biotin-conjugated maleimide. Labeling with this reagent inhibited the binding of cytochalasin B to the transporter. Following sodium dodecyl sulfate-gel electrophoresis, labeling of the transporter and its proteolytic fragments was detected by Western blotting and probing with alkaline phosphatase-conjugated avidin. After tryptic cleavage of the transporter into two membrane domains, preparations reacted with galactose oxidase/biotin hydrazide were labeled on the 25-kDa glycosylated fragment, but not on the carbohydrate-free 19-kDa peptide. Biotin-maleimide-labeled cysteine residues on both peptides. Transporter polypeptide was fragmented more extensively using Staphylococcus aureus V8 protease. Limited digestion produced a broad band of 30-50 kDa and sharper bands of 23 and 21 kDa. More extensive digestion resulted in the disappearance of the 23-kDa peptide and the appearance of sharp bands of 20, 19, 17, 13, 11, 8, and 7 kDa. Biotin label introduced with galactose oxidase/biotin hydrazide was found on the broad 30-kDa band, confirming its identity as a glycopeptide. All of the peptides weighing more than 11 kDa contained cysteine residues labeled with biotin maleimide, while the 8- and 7-kDa peptides were unlabeled. These results demonstrate the potential usefulness of biotin-conjugated reagents as site-specific probes of membrane protein structure.     

Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin.

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.     

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.     

Studies with antipeptide antibody suggest the presence of at least two types of glucose transporter in rat brain and adipocyte

Three antipeptide antibodies were prepared by immunizing rabbits with synthesized short peptides corresponding to residues 215-226, 466-479, and 478-492 predicted from the cDNA of both the human hepatoma HepG2 and rat brain glucose transporters. All three antibodies were found to precipitate quantitatively the [3H]cytochalasin B photoaffinity-labeled human erythrocyte glucose transporter. Each antibody also recognized the rat brain protein of Mr 45,000 on immunoblots, and a similar molecular weight protein was labeled with [3H]cytochalasin B in a D-glucose-inhibitable manner, suggesting that this protein is glucose transporter. However, only up to 30% of the labeled rat brain glucose transporters were precipitated, even by repeated rounds of immunoprecipitation. In addition, these antibodies were observed to be unable to immunoprecipitate significantly the [3H]cytochalasin B-labeled rat adipocyte glucose transporter. Further, one-dimensional peptide maps of [3H]cytochalasin B-labeled human erythrocyte and adipocyte glucose transporters generated distinct tryptic fragments. Although Mr 45,000 protein in rat adipocyte low density microsomes was detected on immunoblots and its amount was decreased in insulin-treated cells, the rat adipocyte low density microsomes were much less reactive on immunoblots than the rat brain membranes in spite of the fact that the rat adipocyte low density microsomes contained more [3H]cytochalasin B-labeled glucose transporters. In addition, the ratio of cytochalasin B-labeled glucose transporter per unit HepG2-type glucose transporter mRNA was more than 10-fold higher in rat adipocyte than in rat brain. These results indicate that virtually all the human erythrocyte glucose transporters are of the HepG2 type, whereas this type of glucose transporter constitutes only approximately 30 and 3% of all the glucose transporters present in rat brain and rat adipocyte, respectively; and the rest, of similar molecular weight, is expressed by a different gene.     

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.     

Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection

Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection in order to study the transmembrane disposition of the protein in situ. Proteolytic enzymes as well as glycosidases were used to treat unsealed and resealed ghosts in order to explore the various membrane domains of the transporter in a topographically defined manner. Limited digestion of sealed ghosts with trypsin had no effect on the apparent Mr of the transporter (55,000). Similar treatment in unsealed ghosts, however, resulted in the generation of a major fragment of 21.5 kDa, along with several minor fragments. Thermolysin also had no effect on sealed ghosts but caused a complete loss of radiolabel from the zone 4.5 region with no lower-molecular-weight fragments being retained on the gel. Chymotrypsin treatment resulted in the generation of a single peak, Mr = 18,400, in both sealed and unsealed ghosts indicating its action occurs at the outer surface. Digestion with carboxypeptidase and aminopeptidase indicate the C-terminal end of the transporter is located exterior to the membrane with the N terminus located at the cytoplasmic surface. Treatment with endoglycosidase resulted in a shift of mobility of the transporter to a lower Mr of 49,000. The results obtained indicate that the carbohydrate is located near the C-terminal end and that the cytochalasin B-binding site is located near the cytoplasmic N-terminal end.     

Purification and characterization of the active fragment from Bacillus thuringiensis delta-toxin

Limited tryptic hydrolysis of a partially purified delta-toxin (Mr = 100,000) from Bacillus thuringiensis, has produced a polypeptide fragment of Mr = 60,000 containing the full biological activity. The fragment was the only polypeptide observed in the polyacrylamide-gel electrophoresis of the delta-toxin after treatment with trypsin and could be purified by DEAE-cellulose chromatography. Amino acid and partial sequence analyses indicate that the 60,000 Mr fragment has been derived from the mid-section of the holotoxin peptide; over 80% of Lys, 65% of Pro and 50% of His residues in the holotoxin have been lost in the active fragment. This section must contain the active site since its specific insecticidal activity is approximately twice that of the holotoxin. The active fragment shows complete cross-reactivity with the antiserum raised against the native toxin, and appeared to possess higher thermal stability than the mother protein. It provides a powerful tool for studies of the structure involved in the insecticidal activity.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Amino acid sequence of Escherichia coli biotin carboxyl carrier protein (9100).

The amino acid sequence of a proteolytic fragment of Escherichia coli biotin carboxyl carrier protein was determined from the structures of overlapping tryptic, thermolytic, and staphylococcal protease peptides together with automated sequenator analyses on the intact protein. The fragment, 82 residues in length, contains the single residue of biocytin of the protein. The relationship of the Mr = 9100 fragment to the native Mr = 22,500 subunit is discussed.     

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.