Partial NH2-terminal Amino Acid Research Articles

Bifidin I – A new bacteriocin produced by Bifidobacterium infantis BCRC 14602: Purification and partial amino acid sequence

A three-step purification protocol has been applied for purification of a new bacteriocin, bifidin I, from Bifidobacterium infantis BCRC 14602. The purification protocol resulted in a purification fold of 1390 with a specific activity of 365714 AU mg −1 and a yield of 25.6%. Bifidin I was recovered by adsorption–desorption onto/from silicic acid (ADSA). The adsorption of the bifidin I from B. infantis BCRC 14602 to silicic acid (5%) was strongly affected by the pH of the broth culture of which 100% adsorption occurs between pH 5.0 and 7.0, whereas at pH values below 5.0 and above 7.0, the adsorption ratio decreased to values 67–45% respectively. Mass spectrometry analysis showed the mass of bifidin I to be approximately 2879.7 Da. Only a partial NH 2-terminal amino acid sequence was obtained comprising of 8 amino acid residues; NH 2 – Lys-Tyr-Gly-Ser-Val-Pro-Leu-Gly. The strong antilisterial activity of bifidin I and its ability of inhibiting the growth of many other Gram-positive and Gram-negative bacteria which can cause food spoilage and food borne diseases have great applications in food safety. Curing experiments which resulted in Bif ¯ variants incapable of producing bifidin I but retaining immunity showed that production of bifidin I is plasmid associated.

Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8

Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage.

The Ubiquinone-binding Site in NADH:Ubiquinone Oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.

Identification and purification of proteins from germ cell-conditioned medium (GCCM).

Germ cells are known to regulate Sertoli cell and testicular function possibly through released factor(s) or via cell-cell contact. However, the identities of many of these putative biological factors are not known. The aim of this study is to present a strategy to identify and purify germ cell-derived proteins found in germ cell-conditioned medium (GCCM) at a quantity sufficient to permit protein microsequencing. The purification scheme of a novel germ cell-derived protein from GCCM designated GC-26 is presented along with several germ cell proteins using a combination of high pressure liquid chromatography (HPLC) columns. The purity of GC-26 and other germ cell proteins were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining. The identities of GC-26, a 26-kDa polypeptide, and other proteins were determined by direct protein microsequencing. These partial NH2-terminal amino acid sequences were compared with the existing databases at Protein Identification Resource (PIR), GenBank, and BLAST. These analyses revealed that these proteins are unique. This strategy should be useful for the micropurification of proteins from other biological samples and/or fluids.

Purification and partial sequencing of the major mitogen for human uterine smooth muscle-like cells in leiomyoma extracts.

We purified the major mitogen for human smooth muscle-like cells in leiomyoma extracts by sequential liquid chromatography on (a) carboxymethyl-Sepharose, (b) heparin-Sepharose columns, (c) cartridges of C18 silica, and (d) linear gradient reverse-phase high performance liquid chromatography. The mitogenic activity of the leiomyoma extract throughout purification was tested by tritiated thymidine incorporation and DNA content in NIH/3T3 fibroblasts and KW human smooth muscle-like cells. Purification of the leiomyoma-derived growth factor (LDGF) for KW smooth muscle-like cells confirmed that its partial NH2-terminal amino acid (aa) sequence (1-20 aa) was identical to 113-132 aa of the human cysteine-rich protein (hCRP). A synthetic peptide which was engineered based on the purified aa sequence, stimulated the proliferation and growth of KW cells. An oligonucleotide probe constructed by the cDNA of the hcrp gene that encodes this aa sequence depicted the expression of 1.9-kb LDGF mRNA in leiomyomas and myometrium. The expression of the LDGF mRNA was three to sixfold higher in leiomyomas compared with adjacent myometrium of women harboring leiomyomas by in situ hybridization analysis. These data suggest that LDGF may participate in the pathophysiology of uterine leiomyomas.

Purification of a candidate gonadotropin surge inhibiting factor from porcine follicular fluid.

Several lines of evidence suggest that the ovaries of many species produce a nonsteroidal substance, termed gonadotropin surge inhibiting factor (GnSIF), which inhibits the midcycle gonadotropin surge and attenuates the pituitary response to endogenous or exogenous GnRH. We have previously reported the partial purification of GnSIF from porcine follicular fluid (pFF) and its differentiation from inhibin. We present now the purification of GnSIF to homogeneity and determination of the partial NH2-terminal amino acid sequence. The bioassay for GnSIF used rat pituitary cells in short-term culture that were incubated with test fractions for 48 h, washed, and then incubated with 10 nM GnRH plus test fractions for 4 h. GnSIF activity is defined as the suppression of GnRH-stimulated LH secretion. GnSIF was purified from 500 ml of pFF using sequential heparin-Sepharose, anion-exchange, and cation-exchange liquid chromatography followed by gel permeation, hydrophobic interaction, and mono-Q HPLC steps. Using these six purification steps, we have obtained an apparently homogenous preparation that stains as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GnSIF has an apparent mol wt of 69K. Limited NH2-terminal sequence analysis reveals that GnSIF has no sequence homology with other reproductive hormones including the inhibins, activins, and follistatins. Over the dose range tested, GnSIF had no effect on basal LH or FSH secretion by pituitary cells in culture and only slightly inhibited GnRH-stimulated FSH secretion at the highest dose tested. In addition, there was no inhibin or follistatin immunoactivity in the GnSIF preparation. As such, GnSIF appears to be a novel protein in pFF that inhibits GnRH-stimulated LH secretion, and which may participate along with other ovarian proteins and steroids in the regulation of pituitary gonadotropin secretion.

The Isolation and Comparison of Multiple Forms of CYP2B from Untreated and Phenobarbital-Treated Rabbit Liver Microsomes

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 12 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1. was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physicochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.

Isolation of antimicrobial peptides from avian heterophils

Five bactericidal peptides (chicken heterophil peptides CHP1 and CHP2; turkey heterophil peptides THP1, THP2, and THP3) were purified from avian heterophil granules. All peptides were cationic and rich in cysteine, arginine, and lysine. The complete amino acid sequence, consisting of 39 amino acids, was determined for CHP1. This peptide had a molecular weight of 4481 as determined by mass spectrometry. Partial NH2-terminal amino acid sequences were obtained for the remaining peptides. Both chicken peptides and THP1 shared sequence homology at 22 residues and a cysteine motif which was similar to that of bovine beta-defensins. THP2 and THP3 were homologous to each other but were not homologous to the other three and had a unique cysteine motif. Peptides CHP1, CHP2, and THP1 killed Staphylococcus aureus and Escherichia coli in vitro, whereas THP2 and THP3 killed only S. aureus in vitro.

The biosynthesis of molybdopterin in Escherichia coli. Purification and characterization of the converting factor

Molybdopterin, the universal component of the pterin molybdenum cofactors, contains a dithiolene group serving to bind Mo. Addition of the dithiolene sulfurs to a molybdopterin precursor requires the activity of the converting factor. Active converting factor has been purified from Escherichia coli chlA1 cells and found to have two subunits of mass 10 and 16 kDa. Electrophoresis of the purified converting factor on denaturing polyacrylamide gels revealed the presence of a 27-kDa protein as well. Partial NH2-terminal amino acid sequencing showed that the 27-kDa protein is a covalent complex of the 10- and 16-kDa proteins. The inactive converting factor purified from E. coli chlN contains both subunits, as established by amino acid sequencing of the purified material, but the 10-kDa subunit is inactive. Absence of the 27-kDa complex in chlN preparations showed that the inactive covalent complex is only formed from the active converting factor. Evidence that activation of the small subunit requires the acquisition of sulfur was obtained from the difference in the molecular masses of the 10-kDa proteins isolated from chlA1 and chlN cells and from the differential sensitivities of the chlA1 and chlN converting factors to iodoacetamide.

Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.

Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid.

The amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predominant amino acids in the phospholipase A2 were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu- Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the "Group I" enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.

Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase

Core RNA polymerase and several forms of RNA polymerase holoenzyme from Bacillus subtilis are found in association with a 21500-kDa polypeptide called delta (δ). We have cloned the structural gene ( rpoE) for δ by using as a hybridization probe a synthetic oligodeoxynucleotide that was designed on the basis of a partial NH 2-terminal amino acid (aa) sequence of purified δ protein. The rpoE gene was found to encode a 173-aa polypeptide of a predicted M r of 20 400. Genetic and physical mapping experiments placed rpoE at 325° on the B. subtilis chromosome and established the gene order rpoE-ctrA-spoOF. The δ subunit is known to enhance the specificity of transcription in vitro by bacterial and phage SP01-modified forms of polymerase, but replacement of rpoE by an in vitro-constructed deletion-mutated gene was found not to impair viability, sporulation, or the growth of phage SP01.

Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.

We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.

Molecular characterization of a subtype of DQw1 recognized by hapten-specific T cells.

Previous studies of HLA-restricted antigen recognition by cloned T cells have frequently demonstrated reactivity that did not correlate precisely with the expression of serologically defined HLA specificities. To further explore such discrepancies, we utilized monoclonal antibody (MoAb) blocking, partial NH2-terminal amino acid sequencing, and Southern blot hybridization techniques to analyze the fine specificity of four autologous trinitrophenyl-specific T cell lines restricted to DR2-linked epitopes. MoAb blocking studies demonstrated that two of these lines recognized determinants on DR molecules while the other two recognized determinants on the same molecule that expresses the DQw1 determinant. However, these latter two lines appeared to recognize a DQw1-related determinant found primarily in association with DR2, but not the other DQw1-associated DR alleles, DR1 and DRw6. To ascertain whether these lines were defining a functional split of DQw1, we performed partial NH2-terminal amino acid sequencing of the molecules precipitated with a DQw1-specific MoAb (Genox 3.53) from different stimulator lines. The results showed that these T cell lines recognized a subtype of DQw1 that is in linkage disequilibrium with DR2. Moreover, we identified characteristic restriction fragment length polymorphisms with a DQ beta-specific cDNA that correlated with stimulatory capacity for the DQw1-restricted lines. These results demonstrate that: DQ molecules may provide restriction determinants that are incorrectly assigned to DR molecules on stimulator panel analyses; cloned antigen-specific T cell lines recognize polymorphic regions of class II molecules not distinguished by either conventional typing antisera or xenogeneic MoAb; and the DQw1 epitope(s) is located on a heterogeneous group of DQ molecules that differ from each other in the primary sequence of their beta chains.

Protein-chemical analysis of pertussis toxin reveals homology between the subunits S 2 and S 3, between S 1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S 2 as the haptoglobin-binding subunit

The purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S 1, free S 5 and the free complexes S 2-S 4 and S 3-S 4, with S 2-S 4 as the only haptoglobin-binding moiety, identifying S 2 as the haptoglobin-binding protein. Partial NH 2-terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS-polyacrylamide gel electrophoresis followed by electroblotting onto polybrene-coated glass-fiber sheets. The sequences reveal extensive homology of the N-terminal portions of the constitutive subunits S 2 and S 3 and between S 1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli.

Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line.

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.

Purification and partial sequence analysis of human interleukin-2 derived from peripheral blood leukocytes

Human peripheral blood leukocyte-derived interleukin-2 (IL-2) was resolved by DEAE-cellulose column chromatography into three peaks of activity, IL-2A, B, and C, with isoelectric points of 7.2, 6.6, and 7.9, respectively. IL-2 A, B, and C were further purified by reverse phase high performance liquid chromatography and resolved into two apparently homogeneous peaks each with identical molecular weight: A-1 and A-2 (Mr17000); B-1 and B-2 (Mr17500); and C-1 and C-2 (Mr14400). The amino acid compositions and partial NH2-terminal amino acid sequences of these molecular species were consistent with those predicted from IL-2 cDNA sequences derived from Jurkat and peripheral blood leukocytes.

Amino acid sequence analysis of the H-2K k alloantigen: Complete sequence of residues 1–98 and partial sequence from 99 to 263

The H-2K k molecule was purified by immunoprecipitation from the glycoprotein fraction of Nonidet P-40 extracts of RDM 4 mouse tumor cells. Cyanogen bromide cleavage of the major papain fragment yielded three peptides, the largest of which consisted of three disulfide-linked peptides which could be separated after reduction and alkylation. These peptides were readily aligned by their homology to similar fragments derived from other H-2 class I molecules. Amino acid sequence analyses of the two nondisulfide-linked peptides, peptide E (residues 1–52) and peptide D (53–98), yielded the following NH 2-terminal sequence for the H-2K k molecule: G-P-H-S-L-R-Y-F-H-T-A-V-S-R-P-G-L-G-K-P-R-F-I- S-V-G-Y-V-D-D-T-Q-F-V-R-F-D-S-D-A-E-N-P-R-Y-E-P-R-(A)-R-(W)-(M)-E-Q-V-E-P-E-Y-W-E-R- N-T-Q-I-A-K-G-N-E-Q-I-F-R-V-N-L-R-T-A-L-R-Y-Y-(N)-Q-S-A-G-G-S-H-T-F-Q-R-(M). Comparison of this sequence with those of other H-2 class I molecules revealed that: (1) Lys-19, Val-55, Glu-56, Asn-63 and Ile-73 are unique to the H-2K k molecule; and (2) H-2K k shares 79–83% homology in this region with other mouse class I molecules. Partial NH 2-terminal amino acid sequences are also reported for the three disulfide-linked peptides. Several discrepancies from previously reported partial sequences of the H-2K k molecule were detected.

Characterization of pepsin-resistant peptides present in a glycoprotein isolated from lung lavage of normal rabbit

A glycoprotein of M r 36000 has been isolated from lung lavage of normal rabbit and purified to homogeneity by gel chromatography. Three peptides containing hydroxyproline and nearly 30% glycine have been isolated and purified from pepsin-digested native glycoprotein. Partial NH 2-terminal amino acid sequence analysis on one of the peptides indicated the presence of -Gly-Pro-Hyp-Gly- sequence in the peptide chain, suggesting that collagen-like region(s) may be present in this glycoprotein.