A radioimmunoassay specific for [Gln]LH-RH: Application in the confirmation of the structure of chicken hypothalamic luteinizing hormone-releasing hormone

In the first report on the chemical structure of a nonmammalian LH-RH, chicken hypothalamic LH-RH was demonstrated to be [Gln⁸]LH-RH [2–4]. However, these studies and subsequent reports [7,8] did not totally exclude the possibility of a reverse sequence of the two amino acids Leu-Gln. In view of the recently described structure of salmon brain LH-RH as [Trp⁷,Leu⁸]LH-RH [9], we undertook to confirm our earlier conclusion that chicken LH-RH is [Gln⁸]LH-RH and not [Gln⁷,Leu⁸]LH-RH. The immunologic, chromatographic and biological properties of natural chicken hypothalamic LH-RH were compared with those of the two synthetic peptides, [Gln⁸]LH-RH and [Gln⁷,Leu⁸]LH-RH. A radioimmunoassay highly specific for [Gln⁸]LH-RH was developed. Natural chicken LH-RH cross-reacted fully with the antiserum which requires the COOH-terminal Gln⁸ to Gly¹⁰-NH₂ for binding, while [Gln⁷,Leu⁸]LH-RH showed less than 0.1% cross-reaction. On a high resolution reverse phase high performance liquid chromatography system, natural chicken LH-RH co-eluted with [Gln⁸]LH-RH and was well separated from [Gln⁷,Leu⁸]LH-RH. In a chicken anterior pituitary cell bioassay, natural chicken LH-RH and [Gln⁸]LH-RH were equipotent in stimulating luteinizing hormone release, while the relative potency of [Gln⁷,Leu⁸]LH-RH was 4.4%. These data, in particular the use of a specific [Gln⁸]LH-RH antiserum, provide conclusive evidence that chicken LH-RH is [Gln⁸]LH-RH.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His⁵,Trp⁷,Tyr⁸-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp⁷,Leu⁸-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln⁸-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

Antitumor effects, cell selectivity and structure-activity relationship of a novel antimicrobial peptide polybia-MPI

A novel antimicrobial peptide, polybia-MPI, was purified from the venom of the social wasp Polybia paulista. It has potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, but causing no hemolysis to rat erythrocytes. To date, there is no report about its antitumor effects on any tumor cell lines. In this study we synthesized polybia-MPI and studied its antitumor efficacy and cell selectivity. Our results revealed that polybia-MPI exerts cytotoxic and antiproliferative efficacy by pore formation. It can selectively inhibit the proliferation of prostate and bladder cancer cells, but has lower cytotoxicity to normal murine fibroblasts. In addition, to investigate the structure–activity relationship of polybia-MPI, three analogs in which Leu⁷, Ala⁸ or Asp⁹ replaced by l-Pro were designed and synthesized. l-Pro substitution of Leu⁷ or Asp⁹ significantly reduces the content of -helix conformation, and l-Pro substitution of Ala⁸ can disrupt the -helix conformation thoroughly. The l-Pro substitution induces a significant reduction of antitumor activity, indicating that the -helix conformation of polybia-MPI is important for its antitumor activity. In summary, polybia-MPI may offer a novel therapeutic strategy in the treatment of prostate cancer and bladder cancer, considering its relatively lower cytoxicity to normal cells.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

A novel metalloprotease from Bacillus cereus for protein fibre processing

A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn³, Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴, Glu²¹, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P₁ position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K_m values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.     

Degradation of cholecystokinin octapeptide by the neutral endopeptidase EC 3.4.24.11 and design of proteolysis-resistant analogues of the peptide

Inactivation of cholecystokinin octapeptide in vitro involves a metalloendopeptidase (EC 3.4.24.11) also called enkephalinase that inactivated the peptide both by a sequential pathway of hydrolysis (removal of Phe-NH₂ followed by cleavage of Trp-Met-Asp) and by an endopeptidase action (production of the tetrapeptides).

As enkephalinase cleaved CCK-8 at the Gly⁴-Trp⁵, Trp⁵-Met⁶ and Asp⁷-Phe⁸ bonds, we investigated the stability of analogues having: (1) substitutions of amino acids by a stereoisomer, (2) a substitution of Asp⁷ by a β Ala residue and (3) modifications of the Trp residue obtained by replacing the nitrogen atom in the indol ring by either an oxygen ([Bfa⁵]CCK-8) or a sulphur atom ([Bta⁵]CCK-8). Among these different CCK derivatives, [βAla⁷], [ Met⁶] and [ Trp⁵]CCK-8 were not hydrolyzed by enkephalinase: [ Ala^d]CCK-8 was rapidly cleaved by the enzyme. [Bta⁵] and [Bfa⁵]CCK-8 did not prove to be quite resistant; however the C-terminal tetrapeptides having the same modifications on the Trp residue were not cleaved although they interacted with the enzyme binding site. The stability and biological activity of the peptidase-resistant analogues of CCK-8 remain to be determined in vivo.     

Structure-activity relationships of trout bradykinin ([Arg(0),Trp(5),Leu(8)]-bradykinin)

Trout bradykinin ([Arg⁰,Trp⁵,Leu⁸]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala⁴, D-Pro³, D-Trp⁵, D-Ser⁶, and D-Pro⁷ substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala⁵] was a weak partial agonist. The substitution (Arg⁰ → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe⁸ in both BK and desArg⁹-BK in activating the mammalian B₂ and B₁ receptors respectively, substitutions at Leu⁸ in trout BK had only a minor effect on potency. Antagonists to the mammalian B₂ receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg⁰,Trp⁵,D-Phe⁷,Leu⁸]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B₂ receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg⁰ in the peptide and an acidic residue in the receptor.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity

Abstract Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser²²⁷, Ser²³⁰ and Ser³⁰⁹ were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser²³⁰ had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser²²⁷→Cys had no effect, Ser²²⁷→Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser³⁰⁹→Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser³⁰⁹→Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser³⁰⁹ is involved in substrate acylation.     

The enzyme stability of dehydro-enkephalins

Dehydro-enkephalins [ΔAla²]-, [ΔAla³]-, [ΔPhe⁴]-, and [ΔLeu⁵]enkephalins, were examined for their stability to enzymatic hydrolysis by carboxypeptidase Y [EC 3.4.16.1]. The successively liberated amino acids were determined quantitatively by amino acid analyses. The saturated leucine-enkephalin was rapidly hydrolyzed from the COOH-terminus. However, peptide linkages with ,β-dehydroamino acid residues placed in the enkephalin molecule were strongly resistant to the enzyme at the carboxyl side and completely resistant at the amino side of the dehydro residue.     

Structure-activity relationship in vasoconstrictor effects of sarafotoxins andendothelin-1

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin-1 (ET-1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET-1> SRTb> SRTa> SRTc at lower doses, but SRTb> ET-1> SRTa> SRTc at higher doses. [Nitrophenylsulfenylated Trp²¹]SRTb and SRTb(1-19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non-natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp²¹ and the intramolecular loop structure are essential, and Lys⁹ and Tyr¹³ may play some important roles for the vasoconstrictor activity of these peptides.     

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between the surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) method in a structural analysis of human endothelin-1

In this study, an automated surface plasmon resonance (SPR)-based biosensor was compared with a quartz crystal microbalance (QCM) biosensor. The two biosensor systems were used for characterizing a site-directed monoclonal antibody (mAb), raised against the C-terminal heptapeptide ET-1_15–21 of the human endothelin (ET-1). The mAb was characterized by its capacity for binding to ET-1, ET-3, Big.ET-1_22–38, the C-terminal (ET-1_15–21, ET-1_16–21, ET-1_17–21), and six derivates of ET-1_16–21, each containing a substitution with alanine (Ala) of a single aminoacid from position 16–21, respectively. The mAb reacted well with ET-1 and its fragments ET-1_15–21, ET-1_16–21, ET-1_17–21, but showed only a partial cross-reaction with ET-3, and did not bind human Big.ET-1_22–38. The Ala substitution on position 16,17, or 19 of ET-1_16–21 did not affect the antibody binding capacity of the hexapaptide ET-1_16–21. On the contrary, Ala substitution or Asp¹⁸, Ile²⁰ and particularly Trp²¹, inhibited its immunoreactivity. Thus the C-terminal represents an immunodominant epitope in ET-1 and is important for antibody binding. The SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, we found a good correlation between results obtained using the BIA technology and the QCM.     

Parathyroid hormone binding sites in the brain

Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of ¹²⁵I-labeled [Nle^8,18,Tyr³⁴]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle^8,18,Tyr³⁴]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, B_max = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system.     

Anaerobic breakdown of uroporpophysrins I and II to bile pigments by extracts of Clostridium tetanomorphum

Abstract The five conserved tryptophan residues in the cellulose binding domain of xylanase A from Pseudomonas fluorescens subsp. cellulosa were replaced with alanine and phenylalanine. The mutated domains were fused to mature alkaline phosphatase, and the capacity of the hybrid proteins to bind cellulose was assessed. Alanine substitution of the tryptophan residues, in general, resulted in a significant decrease in the capacity of the cellulose binding domains to bind cellulose. Mutant domains containing phenylalanine substitution retained some affinity for cellulose. The C-terminal proximal tryptophan did not play an important role in ligand binding, while Trp¹³, Trp³⁴ and Trp³⁸ were essential for the cellulose binding domain to retain cellulose binding capacity. Data presented in this study suggest major differences in the mechanism of cellulose attachment between Pseudomonas and Cellulomonas cellulose binding domains.     

Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca²⁺ in the complex, and shifted the optimum Cl⁻ concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl⁻ concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl⁻.