Biochemical comparison of pili from variants of Neisseria gonorrhoeae P9

Four different types of pili produced by variants of Neisseria gonorrhoeae P9 were isolated and characterized. The pili differed in subunit molecular weight with SDS-PAGE and in subunit isoelectric point on agarose gels. Isoelectric points of the major molecular species in Triton-agarose gels of octylglucoside solubilized pili were: delta, pI 6.5; alpha, pI 6.0; beta, pI 5.3 and gamma, pI 5.5. Amino acid analyses of pili showed close homology between different types but a reduction in the content of aspartate and serine was notable in the low molecular weight delta pili; also beta and gamma maps of tryptic/chymotryptic digests of pili with several major peptides apparently common to all four pilus types.     

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.     

Amino acid substitution and chemical characterization of a Japanese variant of carbonic anhydrase I: CA I Hiroshima-1 (86 Asp → Gly)

An electrophoretic variant of red cell carbonic anhydrase I, designated CA I Hiroshima-1, has been observed in 12 apparently unrelated individuals during a survey of 13,019 individuals from the cities of Hiroshima and Nagasaki, Japan. Analyses of tryptic and chymotryptic peptide patterns of this CA I variant purified from 8 of the 12 individuals revealed the same altered peptides in each case. Examination of the amino acid sequence of an altered tryptic peptide purified from one of the variants showed that the aspartic acid residue at position 86 was replaced by a glycine residue. Thermostability studies demonstrated that all samples of CA I Hiroshima-1 were less stable than normal CA I. The specific esterase (p-nitrophenyl acetate) activities of the normal and variant CA I isozymes were essentially the same. The difference spectra of the normal and variant enzymes were essentially the same. The isoelectric focusing patterns of CA I Hiroshima-1 showed a different pattern of minor bands to those produced by normal CA I. The relative amounts of the normal and variant enzymes purified from single heterozygous individuals were similar.This work was supported by U.S. Public Health Service grant GM-24681 and U.S. Department of Energy contract 2828 (to Dr. J. V. Neel).     

Microheterogeneity of biliverdin reductase in rat liver and spleen: selective suppression of enzyme variants in liver by bromobenzene

Recently we have reported the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat liver. We now report an apparent selective change in the electrophoretic profile of the reductase variants in the liver by in vivo bromobenzene treatment (2 mmol/kg, sc, 24 h). Using two-dimensional electrophoresis and isoelectric focusing, one molecular mass species of the reductase (Mr 30,400) appeared to be selectively suppressed by bromobenzene treatment. This molecular mass species was the main component of two isoelectric focusing bands with pI6.23 and 5.91. The effect in vivo of bromobenzene could not be duplicated by in vitro experiments involving treatment of purified enzyme with bromobenzene in the presence of a NADPH-dependent microsomal drug metabolizing system. The phenomenon of multiplicity of the reductase was not limited to the liver. Multiplicity of the enzyme was detected also in the spleen; however, the pattern of composition of the reductase variants vastly differed from that of the liver. In the spleen, variants with pI 5.76, 5.61, and 5.48 were the prevalent forms; the variant with pI 6.23 was absent, and pI 5.91 was present in a minute amount. Further, bromobenzene did not affect the composition pattern of net-charge variants in this organ. Also, the splenic biliverdin reductase activity was refractory to in vivo bromobenzene treatment, whereas the liver reductase activity with both NADH and NADPH was altered by the treatment. The possible significance of the presence of multiple variants of biliverdin reductase and the change in their composition caused by bromobenzene is discussed.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.     

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.     

Detecting low level sequence variantsin recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5–5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274Tat 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.Key words: recombinant monoclonal antibody, cell line development, sequence variants, HPLC-UV/MS/MS, tryptic peptide mapping, Mascot error tolerant search     

Identification in turkey gizzard of an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase

The isolation of an acidic protein, pI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is concluded that this protein is closely related to the C-terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C-terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed "telokin" (from a combination of kinase and the Greek telos, "end").     

A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues--pancreatic, lacrimal, and submandibular--from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping

The catalytic alpha-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody ("9-A5") covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump's alpha-subunit from [gamma-32P]ATP [Schenk, D. B., Hubert, J.J., & Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renal alpha-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mr 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal beta-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic "beta"-subunits (Mr 50K and 55K) eluted from 9-A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50 = 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike alpha form) and, if at all, structurally divergent forms of the sodium pump's beta-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps.     

Further characterization of the major forms of reptile beta-endorphin

Biosynthetically labeled reptile intermediate pituitary beta-endorphin-sized material was fractionated by SP-Sephadex ion exchange chromatography into two major opiate-active forms which eluted at 0.28 M NaCl and 0.32 M NaCl, respectively; the 0.32 M form of reptile β-endorphin (mw=3500), serves as the precursor for the 0.28 M form of reptile β-endorphin (mw=3200), (Dores and Surprenant, 1983). Analysis of tryptic digests of these reptile β-endorphins by paper electrophoresis at pH 3.5 and gel filtration on a Sephadex G-15 column indicated that there are two tyrosine residues, two arginine residues and one methionine residue in reptile β-endorphin. Furthermore, the NH₂-terminal tryptic peptide of both reptile β-endorphins is approximately nine amino acids in size and contains tyrosine, methionine and arginine. Analyses of chymotryptic/protease digests of the [³H]tyrosine-labeled NH₂-terminal tryptic peptide analyzed by descending paper chromatography revealed that the NH₂-terminal tyrosine of reptile β-endorphin is not -N-acetylated. A second tyrosine-containing tryptic peptide was detected in the COOH-terminal region of reptile β-endorphin; however this tryptic peptide differs in the two forms of reptile β-endorphin in terms of size and net charge at pH 3.5. These differences account for the apparent molecular weight differences and distinct ion exchange properties of the 0.28 M and 0.32 M forms of reptile β-endorphin. Thus in the reptile intermediate pituitary the principal post-translational mechanism for modifying β-endorphin is COOH-terminal proteolytic cleavage.     

Analysis of the molecular species of the chick oviduct progesterone receptor using isoelectric focusing.

Conditions are described for the preparative isoelectric focusing in flat beds of Sephadex of the progesterone receptor from the chick oviduct. The method allows the fractionation of the receptor into two molecular species, one focusing at pI 6 and the other at pI 7 with good purification and recovery. The pI 6 and pI 7 receptor species were purified 2- and 26-fold, respectively. The assaying of the focused fractions with the charcoal binding method provides an accurate identification and quantitation of the [3H]progesterone receptor. The method is reproducible in recovery, quantitation, and resolution of the two receptor species. The receptor with an apparent pI of 6 sediments at approximately 4 S on linear sucrose gradients, while the receptor with an apparent pI of 7 sediments at approximately 3.5 S. On the basis of the sedimentation values and elution patterns from diethylaminoethyl (DEAE) chromatography, the pI 6 component is equivalent to the "B" receptor species and the pI 7 component is equivalent to the "A" receptor species described previously [schrader, W, T., 7 O'Malley, B. W. )1972) J. Biol. Chem. 241, 51--59].     

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.     

Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing

The venom from the scorpion, Centruroides sculpturatus Ewing (range, Southwestern United States), was fractionated into ten protein zones by chromatography on CM-cellulose. Further purification of three of these zones on DEAE Sephadex in ammonium acetate developers yielded three principal neurotoxins designated variants 1, 2, and 3. Variants 1 and 3 each consist of 65 amino acid residues, variant 2 is composed of 66 residues, and each variant is a single polypeptide chain crosslinked by four disulfide bridges. The three variants have lysine at the amino terminus, serine at the carboxyl terminus, and their sequences exhibit a high degree of homology. The complete structure of variant 2 was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. The sequences of most of the tryptic peptides of variants 1 and 3 were determined, and the peptides were aligned by comparison with the homologous peptides in variant 2. The results show that there are 4 differences in sequence between variants 2 and 3 and 9 differences between variants 1 and 2. Variants 1 and 3 differ from each other at 5 positions in their sequences. These differences between the three protein variants are found in the amino terminal one-third of the molecules except for the deletion of one seryl residue at the carboxyl terminal of variants 1 and 3.     

Operation of the glucose-fatty acid cycle after glycogenolysis induced by treatment with nicotinic acid.

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work (J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.     

Detecting low level sequence variants in recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5-5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274T at 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.     

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.     

The amino acid sequence of the ribosomal protein S8 of Escherichia coli.

The primary structure of protein S8 from the 30S subunit of Escherichia coli ribosomes has been determined by sequencing the peptides derived from tryptic, chymotryptic, thermolytic and staphylococcal protease digestion of the protein. Protein S8 has 129 amino acid residues which result in a molecular weight of 13996. The N-terminal part of the sequence up to position 68 is in complete agreement with the reported sequence data[1,2]. However, differences exist in the C-terminal half, where an additional hydrophobic tryptic peptide has been found.