Production of biologically active fragments of parathyroid hormone by isolated Kupffer cells

Cleavage of parathyroid hormone (PTH) by isolated Kupffer cells from rat liver was examined. Iodinated PTH labeled at position 43 was converted into two radioactive fragments which were shown by Edman degradation to have residues 35 and 38 as their NH2 termini. Cleavage at these positions is characteristic of cathepsin D. Amino-terminal fragments were detected by bioassay of fractions obtained by high performance liquid chromatography. These fragments eluted in positions characteristic of the 1-34 and 1-37 peptides also previously shown to be produced by purified cathepsin D. The putative 1-37 fragment was rapidly converted to 1-34 upon digestion with cathepsin D, whereas the putative 1-34 fragment was not further digested by this enzyme, behavior previously shown to be characteristic of 1-37 and 1-34 bovine PTH. Fragmentation of PTH as measured by generation of fragments soluble in trichloroacetic acid was inhibited by methylamine, monensin, and ammonium chloride. In addition, monensin significantly inhibited production of both carboxyl- and amino-terminal fragments. Finally, active PTH fragments were also produced by elicited peritoneal macrophages. It is concluded that Kupffer cells, and other macrophages, can produce active fragments of PTH which appear in the medium. These fragments may be generated by cathepsin D within the cells.     

Formation and secretion of fragments of parathormone. Identification of cleavage sites

Monolayer cultures of bovine parathyroid cells or fresh gland slices were incubated with radioactive amino acids in order to study the formation and metabolism of parathormone (PTH). PTH, secretory protein I, and COOH-terminal fragments of PTH were all released into media within 30 min, most strongly in the first hour after synthesis. Peptides in tissue, cells, and media were separated using reverse-phase high performance liquid chromatography. In eluates of media, six radioactive peaks were prominent. The first four and the sixth were immunoreactive in a COOH-terminal specific PTH radioimmunoassay, but only the sixth was reactive in an NH2-terminal specific assay. Under conditions where recovery of PTH(1-34) was quantitative, gel filtration of media was used to show that no NH2-terminal fragments of PTH were secreted. Sequence analyses of secreted COOH-terminal peptides indicated that the NH2 termini of the first three peaks corresponded to residues 43, 37, and 34 of PTH. The fourth peak contained a mixture of two peptides with NH2 termini at residues 24 and 28 of PTH. The fifth could not be identified; the sixth was PTH. Cleavages at the 23-24 bond of PTH occurred within minutes of the formation of PTH itself, and the other peptides were formed more slowly. Mandatory cleavage of PTH at the 23-24 peptide bond would destroy the biological activity of the hormone on kidney and bone, a situation consistent with the possibility that intracellular PTH metabolism participates in secretory regulation. The results showed that different peptides were generated in parathyroid cells than were previously shown to be produced by cathepsin B or D. The results suggest that the proteolytic pathway which results in the secretion of PTH fragments is nonlysosomal in nature.     

The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B.

Purified cathepsin B from porcine parathyroid glands was allowed to act upon radioactive bovine parathormone and proparathormone at various ratios of enzyme to substrate and for different times. The reaction products were isolated by ion exchange chromatography and analyzed by gel electrophoresis, amino acid composition, sequence analysis, and bioassay. The enzyme cleaved parathormone between residues 36 and 37 yielding a major carboxyl and amino fragment and appeared to cleave proparathormone at the same locus. The amino fragments were degraded further by removal of small peptides (possibly, di- or tripeptides) from their COOH termini. In contrast there was little if any degradation of the carboxyl fragment (residues 37 to 84). Despite the ease with which the enzyme cleaved the arginyl bond in the synthetic substrate benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide, it did not remove the near homologous NH2-terminal hexapeptide extension of proparathormone (Lys-Ser-Val-Lys-Lys-Arg-R)--a reaction that would lead to the formation of parathormone from proparathormone. Purified liver cathepsin B cleaved the hormonal substrates in a fashion identical with that of the parathyroid enzyme.     

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2- terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.     

Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37)

Parathyroid hormone (PTH) is involved in regulation of the calcium level in blood and has an influence on bone metabolism, thus playing a role in osteoporosis therapy. In this study, the structures of the human PTH fragments (1-34) and (1-39) as well as bovine PTH(1-37) in aqueous buffer solution under near physiological conditions were determined using two-dimensional nuclear magnetic resonance spectroscopy. The overall structure of the first 34 amino acids of these three peptides is virtually identical, exhibiting a short NH(2)-terminal and a longer COOH-terminal helix as well as a defined loop region from His14 to Ser17, stabilized by hydrophobic interactions. bPTH(1-37), which has a higher biological activity, shows a better-defined NH(2)-terminal part. In contrast to NH(2)-terminal truncations, which cause destabilization of helical structure, neither COOH-terminal truncation nor elongation significantly influences the secondary structure. Furthermore, we investigated the structure of hPTH(1-34) in 20% trifluoroethanol solution. In addition to its helix-stabilizing effect, trifluorethanol causes the loss of tertiary hydrophobic interactions.     

Location of a collagen-binding domain in fibronectin

Preferential labeling of COOH-terminal sequences in newly synthesized fibronectin was achieved by short term incorporation of radiolabeled amino acids in the presence of pactamycin, an inhibitor of polypeptide chain initiation. The labeled fibronectin was then cleaved with cathepsin D under conditions that yield a large (137,000-dalton) fragment that lacks collagen-binding properties, and a smaller (72,000-dalton) fragment that retains the ability of fibronectin to bind to collagen. Determination of the relative specific radioactivities of the two fragments leads us to conclude that the collagen-binding domain in fibronectin is located in the NH2-terminal third of the polypeptide chain and not in a COOH-terminal region as previously indicated by other structural studies.     

Interaction of the Two Structural Domains of Calmodulin with Mature and Immature Rat Brain Microtubules

The inhibitory effect of calmodulin on the assembly of mature and immature rat brain microtubules was compared with that of the two major structural domains of this protein, the COOH-terminal fragment (amino acids 78-148) and the NH2-terminal fragment (amino acids 1-77), to determine the calmodulin structural domain responsible for the inhibitory effect on microtubule assembly. Microtubules prepared during the early stages of brain development, i.e., during intensive neurite outgrowth, are more sensitive to inhibition by the Ca2(+)-calmodulin complex than those obtained from adult brain. Significant inhibition of immature microtubule assembly was observed with both fragments in the absence of Ca2+, but the effects were more important when Ca2+ was present. With adult brain microtubules, the two fragments remained without effect on assembly in the absence of Ca2+, whereas some inhibition was seen in its presence but only with the COOH-terminal polypeptide. Under all these conditions, the COOH-terminal fragment was always more active than the NH2-terminal fragment on microtubule polymerization, albeit to a lesser extent than native calmodulin.     

NH2-terminal big gastrin immunoreactivity in human urine

The concentrations and molecular forms of urinary and plasma gastrin from normal subjects were studied by radioimmunoassays using two region-specific antisera. Urinary concentration of NH2-terminal big gastrin (G-34) immunoreactivity was several hundred times as great as that of COOH-terminal gastrin immunoreactivity. Fractionation of urine extract showed a broad giant peak of NH2-terminal G-34 immunoreactivity (gastrin fragments "U") eluting in a later position than G-34(1-17) by Sephadex G-50 column chromatography. HPLC revealed that urinary NH2-terminal G-34 immunoreactivity was composed of four fragments including G-34(1-8), G-34(1-9), and G-34(1-10). Sephadex G-50 column chromatography of plasma extract revealed two or three peaks of NH2-terminal G-34 immunoreactivity, and a major peak eluted in the same position as urinary gastrin fragments "U". These results and data on renal clearances suggest that most of all gastrin fragments "U" in plasma are excreted in urine without renal reabsorption, whereas almost all of plasma COOH-terminal gastrin peptides including G-34 and little gastrin (G-17) are removed and metabolized in the kidney.     

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.     

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.     

Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment

By utilizing double-labeled fluorescent cellular prion protein (PrPC), we revealed that the NH2-terminal and COOH-terminal PrPC fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells and HpL3-4, a hippocampal cell line established from prnp gene-ablated mice [Nature 400 (1999) 225]. Of note, the NH2-terminal PrPC fragment, which predominantly localized in the intracellular compartments, congregated in the cytosol after the treatment with a microtubule depolymerizer (nocodazole). Truncated PrPC with the amino acid residues 1-121, 1-111, and 1-91 in mouse (Mo) PrP exhibited a proper distribution profile, whereas those with amino acid residues 1-52 and 1-33 did not. These data indicate the microtubules-associated intracellular localization of the NH2-terminal PrPC fragment containing at least the 1-91 amino acid residues.     

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

Human pituitary growth hormone. 44. Effects of plasmin-modified hormone and its fragments on ornithine decarboxylase activity and lipolysis.

The rat hepatic ornithine decarboxylase stimulating activity of plasmin-modified human growth hormone and its two peptide fragments has been investigated. The activity was completely retained after plasmin treatment. The NH2-terminal fragment [Cys (Cam)53-HGH-(1-134)] retained 10% of the activity, whereas the COOH-terminal fragment [Cys (Cam) 165, 182, 189-(141-191)] was not active. The lipolytic activity of human growth hormone was greatly reduced after plasmin treatment, as examined in isolated rabbit adipocytes. It is suggested that the structural requirements for the lipolytic activity of the hormone are different from those required for stimulation of ornithine decarboxylase activity.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.     

Effect of methionine oxidation and deletion of amino-terminal residues on the conformation of parathyroid hormone. Circular dichroism studies

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors.     

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.