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D A Walz

Publications and source records attributed to D A Walz.

At least 37 records · Page 2Linked to original sources
PubMed1992

Comparison of the hemoglobins of the platyhelminths Gastrothylax crumenifer and Paramphistomum epiclitum (Trematoda: Paramphistomatidae).

1. Gastrothylax crumenifer and Paramphistomum epiclitum parasitize the water buffalo Bubalus bubalis. 2. Gastrothylas hemoglobin consisted of two fractions of ca 30,000 and ca 18,000 by gel filtration. SDS-electrophoresis showed both to be single, ca 15,000 chains. 3. Paramphistomum hemoglobin was ca 16,000 by both gel filtration and SDS-electrophoresis. 4. Reversed-phase chromatography of carboxymethylated trematode and buffalo globins gave single peaks and two peaks, respectively. Although Paramphistomum hemoglobin provided and N-terminal sequence, Gastrothylax hemoglobin did not, suggesting blocked N-terminals. The buffalo sequences were found to be identical to the sequences of the alpha and beta chains of bovine hemoglobin. 5. Although Paramphistomum hemoglobin consists of only one chain, Gastrothylax hemoglobin consists either of one chain which aggregates to a dimer or of two different chains, only one of which aggregates to a dimer.

Amino Acid Sequence↗
PubMed1992

Evidence for the existence of O-linked sugar chains consisting of glucose and xylose in bovine thrombospondin.

We have recently discovered unusual sugar chains [xylose-glucose and (xylose)2-glucose] linked to a serine residue in the first epidermal growth factor (EGF)-like domains of human and bovine coagulation factors VII, IX, and protein Z. The sequence surrounding this serine residue has a common -Cys-X-Ser-X-Pro-Cys- structure. Since one (residues 533-538) of the three EGF-like domains found in human thrombospondin contains the conserved sequence, we examined the presence of such O-linked sugar chains in bovine thrombospondin (bTSP) and its 210-kDa fragment. Component sugar analysis after pyridylamination (PA) of the acid hydrolysates of the S-aminoethylated proteins revealed that the proteins contain glucose (Glc) and xylose (Xyl). The oligosaccharide moieties released from intact bTSP by hydrazinolysis followed by pyridylamination were separated into two PA-oligosaccharides by high performance liquid chromatography (HPLC). Component sugar analysis of these PA-oligosaccharides indicated that they consist of Glc and Xyl in molar ratios of 1:1 and 1:2 (or 1:3). The reducing ends of both PA-sugar chains were found to be PA-Glc, as judged from the retention time of the HPLC peak of their hydrolysates. The presence of these PA-sugar chains in bTSP was confirmed by HPLC mapping with two different columns, using standard PA-di- or PA-trisaccharide derived from coagulation factors. From these results, we concluded that bTSP contains O-linked sugar chains consisting of Glc and Xyl in one of its three EGF-like domains.

Amino Acid Sequence↗
PubMed1991

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

HLA-DR is a procoagulant.

Numerous incidents of thromboembolic complications have been documented in cancer patients and in recipients of mismatched organ transplants. Tumor procoagulants have also been implicated in the process of metastasis. Two protein bands of 35,000 and 28,000 daltons isolated from human ovarian carcinoma possessed procoagulant activity. The 35,000 dalton protein had an amino terminal sequence identical to that of the major histocompatibility antigen HLA-DR. Further, isolation of the protein using immunoaffinity column chromatography with monoclonal antibody to HLA-DR resulted in the isolation of procoagulant activity. The immunoaffinity purified protein enhanced thrombin generation in recalcified normal plasma approximately 20- fold. HLA-DR procoagulant activity was completely inhibited by Staphylococcal enterotoxin A. We propose that the procoagulant nature of HLA-DR may contribute to thrombotic disorders in several cancers and in association with graft rejection. The ability of enterotoxin A to inhibit this procoagulant may lead to development of future therapeutic strategies.

Antibodies, Monoclonal↗
PubMed1991

1H-NMR studies of bovine platelet factor 4: histidine assignments and interactions with heparin.

1H-NMR spectroscopy has been used to assign and to characterize the two histidine C2H resonances of the heparin binding protein, bovine platelet factor 4. One histidine has a pKa value of 6.51 at 27 degrees C; the second histidine exhibits 2 pKa values of 5.52 and 5.66 at 27 degrees C. The two histidine resonances have been assigned by an analysis of their deuterium exchange kinetics. Both resonances are solvent accessible with half-times of exchange at pH 8.8 of 3.3 and 4.0 days. These two resonances have been assigned by digesting partially deuterated protein with Staphylococcus aureus V-8 proteinase, separating and purifying the resulting peptides, and determining their relative and residual hydrogen content by NMR. The results indicate that His-38 has the lower pKa value and the slower deuterium exchange rate, whereas His-50 has the higher pKa value and the faster deuterium exchange rate at pH 8.8. The 1H-NMR resonance of His-38 of bovine platelet factor 4 is preferentially perturbed by the introduction of heparin. This observation and the presence of His-38 within the belt of positively charged residues around the platelet factor 4 tetramer supports the model of the platelet factor 4-heparin complex in which the polysaccharide crosses over each of the 2 alpha-helices of the 2 dimers at right angles.

Animals↗
PubMed1991

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22.

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Chemotactic activity and receptor binding of neutrophil attractant/activation protein-1 (NAP-1) and structurally related host defense cytokines: interaction of NAP-2 with the NAP-1 receptor.

Neutrophil attractant/activation protein-1 (NAP-1) has sequence similarity to platelet factor-4 (PF-4) and to NAP-2 (a truncated from of connective tissue activating protein-III [CTAP-III(des 1-15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP-III, CTAP-III(des 1-13), the C-terminal dodecapeptide of PF-4 [PF-4(59-70)], and C5a. Chemotactic potency (EC50) was highest for NAP-1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP-1, and NAP-2, the NAP-2 response occurred only at concentrations 100-fold higher than the NAP-1 EC50 of 10(8) M. Data for the CTAP-III proteins confirmed that CTAP-III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N-terminus to make CTAP-III(des 1-13) or NAP-2 [CTAP-III(des 1-15)]. Chemotactic activity of PF-4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF-4(59-70) regularly induced high chemotactic responses, although the EC50 of 1.6 x 10(5)M was 1,000-fold greater than that of NAP-1. The binding of fluoresceinated NAP-1 to neutrophils was inhibited by unlabeled NAP-1 or NAP-2 but not by PF-4 or PF-4 (59-70). This suggests that NAP-2 interacts with the neutrophil NAP-1 receptor. Despite the low chemotactic potency of NAP-2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP-III released from platelets, as indicated by a serum concentration of approximately 10(-6) M.

Chemotaxis, Leukocyte↗
PubMed1991

The amino acid sequence of hemoglobin II from the symbiont-harboring clam Lucina pectinata.

The cytoplasmic hemoglobin II from the gill of the clam Lucina pectinata consists of 150 amino acid residues, has a calculated Mm of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J., J. Biol. Chem. 265, 16054-16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of the Lucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 A of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 in Lucina Hb II.

Amino Acid Sequence↗
PubMed1991

Iron and heme contents of the extracellular hemoglobins and chlorocruorins of annelids.

1. A survey of the literature on the extracellular hemoglobins and chlorocruorins of over 30 species of annelids, covering the last 30 years, shows that the range of iron content is 0.211-0.265 wt.% (mean = 0.228 +/- 0.013, N = 28) and the range of the heme content is 1.83-3.64 wt.% (mean = 2.60 +/- 0.38, N = 29). 2. There is relatively little scatter in the values of the experimental iron contents and only one of the 28 values is clearly outside the standard deviation range. 3. The values of heme contents are much more scattered, with seven values, clearly outside the standard deviation limits. 4. The aberrant cases are discussed and it is noted that the mean heme content of 2.60 wt.% corresponds to an iron content of 0.236 wt.% in excellent agreement with the mean iron content of 0.228 wt.%. 5. This result suggests strongly that experimental values of iron and heme contents outside the ranges of 0.211-0.243 and 2.3-2.7 wt.%, respectively, corresponding to a minimum molecular mass outside the range 23,000-26,000, should be regarded with caution.

Animals↗
PubMed1991

Heterogeneity of the subunits of Lumbricus terrestris extracellular hemoglobin dissociated at alkaline pH.

1. The gel filtration profiles of dissociated extracellular hemoglobin of Lumbricus terrestris obtained in 0.1 M borate buffers at pH greater than 9, using columns of Sephadex G100, Sephacryl S200 and Ultragel AcA 44, consist of at least two peaks A and B. 2. SDS-PAGE of several fractions across the complete elution profile demonstrates that only the fractions under the right hand portion of peak B are homogenous and consist of the monomer (M) subunit (Mr = 17,000). 3. The fractions under the first peak contain the remaining subunits, a disulfide-bonded trimer (T) subunit (Mr = 50,000) and of two subunits (D1 and D2) of ca 30,000. 4. Densitometry of the SDS-PAGE patterns suggests that the proportions of these subunits vary across the two peaks, implying that peak A does not consist of a complex of fixed stoichiometry of the T and D1 and D2 subunits. 5. Furthermore, the D1 and D2 subunits overlap the M subunit in the trough between peaks A and B and are present in the left hand portion of peak B, probably because of the self-association of the M subunit. 6. In addition, SDS-PAGE experiments with a single fraction of peak A, where the load and the duration of staining were varied, suggests that the relative proportions of the subunits are independent of these two variables.

Animals↗
PubMed1990

The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216) showed that all five globins have identical residues at 39 positions. At 44 positions, the three polymeric globins share identical residues that differ from the identical residues at the corresponding locations in the monomeric sequences M-II and M-IV including position E7, where the latter have leucine instead of the distal histidine. At 15 positions, there occurs an alteration from polar to nonpolar or from a small nonpolar to a larger nonpolar residue in going from the monomeric to the polymeric globins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex.

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.

Amino Acid Sequence↗
PubMed1990

The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component.

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.

Amino Acid Sequence↗
PubMed1990

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora.

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

A possible receptor-binding function for the N-terminus of connective tissue activating peptide III.

Connective tissue activating peptide III (CTAP-III) is an 85-residue peptide which has been purified from platelets and shown to possess mitogenic activity toward a variety of fibroblastic cell lines. beta-Thromboglobulin (beta TG) is an 81-residue peptide which is derived from CTAP-III by cleavage of the N-terminal tetrapeptide Asn-Leu-Ala-Lys which results in the loss of mitogenic activity. The near-UV CD spectra for the two proteins indicated that the conformations as well as the electronic environments of the two disulfide bonds, and also of the single aromatic tyrosine residue, were similar in CTAP-III and beta TG. However, differences in the far-UV CD spectra of these proteins indicated a substantial decrease in alpha-helical content for beta TG (29%) as compared to CTAP-III (38%). Structure prediction analysis also suggested that the longer N-terminal segment of CTAP-III may form an alpha-helix. The N-terminal region of beta TG, which lacks this tetrapeptide, was predicted to be in an unordered, or possibly a turn, conformation. This predicted structural difference appears to be due to the high helix-forming potential of the N-terminal tetrapeptide Asn-Leu-Ala-Lys in CTAP-III. These results suggest a possible structural role for the N-terminal region of CTAP-III in the expression of the biologic activities of this protein. On the basis of these studies, a reasonable hypothesis to account for the difference in mitogenic activity between beta TG and CTAP-III is that the N-terminal region must be helical for receptor binding to occur.

Amino Acid Sequence↗
PubMed1990

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained.

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Thrombospondin binding to specific sequences within the A alpha- and B beta-chains of fibrinogen.

Thrombospondin is a multifunctional adhesive glycoprotein which binds to the surface of resting and activated platelets. Thrombospondin also binds to a variety of proteins, including fibrinogen. The interactions between platelet-bound thrombospondin and fibrinogen are thought to facilitate irreversible platelet aggregation. Both the A alpha- and B beta-chains of fibrinogen specifically bind to thrombospondin. Cyanogen bromide cleavage products of the fibrinogen A alpha- and B beta-chains, and synthetic peptides corresponding to specific regions of these cleavage products were utilized to identify the regions of the fibrinogen A alpha- and B beta-chains which bind to thrombospondin. Cyanogen bromide fragments of the A alpha- and B beta-fibrinogen chains, resolved by gel filtration and reversed-phase chromatography, were examined for thrombospondin binding activity. Thrombospondin specifically bound to the A alpha-chain fragment encompassing residues 92-147 and the B beta-chain fragment encompassing residues 243-305. Analyses of the binding characteristics of two series of overlapping synthetic peptides revealed that peptides corresponding to residues 113-126 of the A alpha-chain and residues 243-252 of the B beta-chain retained thrombospondin binding activity. Separate bovine serum albumin conjugates of the active A alpha-chain and B beta-chain peptides inhibited platelet aggregation. These studies reveal that fibrinogen possesses at least two unique sequences which are recognized by thrombospondin and that such interaction may affect platelet aggregation.

Amino Acid Sequence↗