Ability of growth hormone fragments to compete with 125I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with ¹²⁵I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with ¹²⁵I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.     

Ability of growth hormone fragments to compete with I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with ¹²⁵I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with ¹²⁵I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.     

Immunochemical studies on big gastrin using NH2-terminal specific antisera

Methods are described for obtaining antisera specific for the NH₂-terminal regions of human and porcine big gastrin (G34) that can be used in radioimmunoassays. Three antisera have been characterized in detail: one (L66) raised to human 1–15 (Tyr⁷Pro⁸Ser⁹) G34 has an antigenic determinant in the 1–6 region of human G34; a second (L107) raised to 1–19 hG34 has an antigenic determinant in the 1–12 region. Both these antisera react weakly with porcine G34. A third antiserum (L33) raised to porcine G34 has an antigenic determinant in the 1–12 region of this peptide, and reacts weakly with human G34. In human antral extracts fractionated on Sephadex G50. L66 and L107 revealed a minor peak of immunoreactivity corresponding to G34, and a major peak corresponding to the NH₂-terminal tryptic peptide of G34. Concentrations of the latter peptide were closely similar to those of G17 (i.e. the COOH-terminal tryptic peptide of G34), consistent with the idea that G34 is cleaved within G-cells by a trypsin-like enzyme to yield G17. Antiserum L33 revealed small amounts of immunoreactivity in antral extracts of dog and cat, but did not reveal significant immunoreactivity in rat antral extracts. In contrast, L66 reacted with rat antral extracts, but not dog or cat. The sequences of G34 in these species are not known, but the results suggest significant differences compared with human and porcine G34, and indicate a high degree of species-specificity with NH₂-terminal G34 antisera.     

Comparative peptide mapping of baculovirus polyhedrins

Amino terminals and two-dimensional high-voltage peptide maps of tryptic digests of polyhedrins from Heliothis armigera nuclear polyhedrosis virus (NPV), Heliothis zea NPV, and Anticarsa gemmatalis NPV were compared with previously characterized granulins and polyhedrins. Similarities and differences were detected in the tryptic maps, while each protein produced a unique peptide map composite. Amino-terminal determination of eight polyhedrins and granulins resulted in three different end groups.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

Revision of the amino acid sequence of human heart fatty acid-binding protein

Summary Cardiac-type fatty acid-binding protein (cFABP) from human heart muscle of three individuals was isolated and characterized as pI 5.3-cFABP. The proteins were structurally analyzed by tryptic peptide mapping, application of plasma desorption time-of-flight mass spectrometry and amino acid sequencing. All three preparations of human heart FABP, having 132 amino acids, differed from the published sequence [Offner et al. Biochem J 251: 191–198, 1988] in position 104, where Leu is found instead of Lys, and in position 124, where Cys is found instead of Ser.     

Bioengineered human growth hormone supports limb regeneration in the hypophysectomized newt Notophthalmus viridescens

It is well documented that growth hormone (GH) replacement therapy will restore normal limb regeneration to hypophysectomized adult newts. However, it is also known that the GH preparations used in previous reports were contaminated by other pituitary hormones shown to support regeneration when administered free of GH. The recent availability of bioengineered human GH was studied for its ability to restore the regenerative capacity to hypophysectomized newts. Five days posthypophysectomy adult newts were subject to forelimb amputation distal to the elbow. Animals were divided into three groups (n greater than 20). Each received one of three GH preparations: pituitary-derived bovine GH, pituitary-derived human GH, or bioengineered human GH. GH was administered via intraperitoneal injection (0.029 IU/50 microliters) on alternate days for either the first 5 days (total of 3 injections) or for 35 days (total of 18 injections). Pituitary-intact and hypophysectomized control newts were subjected to forelimb amputation and injected with hormone diluent. All newts that received GH demonstrated normal limb regeneration to the early digitiform stage by 35 days postamputation. None of the hypophysectomized control newts showed any evidence of regeneration. We conclude that GH alone can restore the ability to undergo normal limb regeneration to hypophysectomized newts.     

Isolation and somatomedin activity of bovine growth hormone fragment 87–124

Various bovine growth hormone (GH) fragements were prepared and tested for somatomedin-like activity in vitro. Cyanogen bromide cleavage followed by reduction and alkylation yielded three fragments which were identified as GH (6–124), GH (150–179) and GH (125–149). No consistent effect was found when these preparations were tested for their ability to stimulate in vitro sulfate and thymidine uptake by rat costal cartilage and to compete with [¹²⁵I]iodoinsulin for insulin-binding sites on placenta membranes. A fourth peptide was isolated by cleavage of the tryptophanyl and methionyl bonds of bovine GH using anhydrous heptofluorobutyric acid and cyanogen bromide. In addition to significant amounts of non-specific cleavage products, a peptide having a molecular weight of about 4800 was isolated. The amino terminal residue was leucine and the carboxyl terminal was homoserine. These data, in addition to the amino acid composition, suggested that the peptide corresponded to residues 87–124. Fragment GH (87–124) stimulated sulfate (minimum effective concentration, 5 · 10^?8 M) and thymidine (minimum effective concentration, 10^?8 M) uptake by rat costal cartilage. It also cross-reacted, albeit weakly, with insulin-binding sites on placenta membrane. Maximum displacement was 35% of non-specific binding. These observations demonstrate that somatomedin-like activity can be generated from the growth hormone molecule which is inherently devoid of such activity.     

Hypophysectomy Prevents Ghrelin-Induced Adiposity and Increases Gastric Ghrelin Secretion in Rats

Objective: The novel gastric hormone ghrelin has recently been identified as an important modulator of energy homeostasis. Leptin-responsive hypothalamic neuropeptide Y/Agouti-related protein neurons are believed to mediate afferent ghrelin signals. Little is known, however, about ghrelin-induced efferent signals. We therefore investigated if hypothalamic-pituitary axes have a role in transferring ghrelin-induced changes of energy balance to the periphery. Research Methods and Procedures: We subcutaneously injected hypophysectomized, as well as adrenalectomized, thyroidectomized, and sham-operated control rats with GH secretagogues [ghrelin, growth hormone (GH)-releasing peptide] for 1 week. Body weight, food intake, and body composition (chemical carcass analysis) were analyzed and compared with vehicle-treated controls. In addition, we quantified circulating levels of endogenous ghrelin in hypophysectomized and GH–treated normal rats. Results: GH-secretagogue treatment of sham-operated control rats dose-proportionally increased food intake, body weight, and fat mass compared with vehicle-injected controls (p < 0.01). These effects, however, were not observed in ghrelin-treated hypophysectomized, thyroidectomized, or adrenalectomized rats, indicating an essential role for the pituitary axis in ghrelin-induced adiposity. Circulating levels of endogenous ghrelin were reduced by administration of GH in normal rats and were about 3-fold higher in hypophysectomized rats (n = 20, p = 0.001), suggesting a regulatory feedback loop involving the stomach and the pituitary to regulate gastric ghrelin secretion. Discussion: According to these results, the endocrine pituitary is mediating ghrelin-induced changes toward a positive energy balance and is involved in the regulation of ghrelin secretion through a gastro-hypophyseal feedback loop.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Protein kinase activity of polysome-ribosome preparations from poliovirus infected cells.

Polysome-ribosome preparations from poliovirus infected HeLa cells contain 10–15 fold more protein kinase activity $\text{in vitro}$ than similar preparations from uninfected cells. Five peptides of mol. wt. 135K, 85K, 65K, 28K and 14K are phosphorylated by polysome-ribosome preparations from infected cells. Extracts obtained from cells infected in the presence of guanidine phosphorylate the same proteins whereas polysome-ribosome extracts obtained from uninfected cells treated with cycloheximide phosphorylate the 135K peptide and two others with mol. wt. values of 77K and 60K. Over 90% of the 135K peptide and over 70% of the other peptides can be washed off ribosomes with 0.5 M KCl.     

Structure and function relationships of three novel hGHRH-GGC analogs

Growth hormone releasing hormone (GHRH) is one of the hypothalamus hormones. For its potential applications in agriculture and medicine, GHRH analog with higher activity and longer half-life has been looked for. By using the fusion expression with unique acid labile linker Asp-Pro and biochemical purification, the three novel GHRH peptides, Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys, Pro-hGHRH(1–44)-Gly-Gly-Cys, and ¹Pro-GHRH(2–44)-Gly-Gly-Cys, were obtained. The peptide molecular weight with 5,455, 5,373 or 5,210 Da measured by EIS-MS is coincident with the actual values. The peptides at 0.1–10 μg/ml increased rat pituitary GH releases in a dose-dependent manner and at 5 μg/ml increased human pituitary GH releases. The activity comparisons showed that at 10 μg/ml there were significant between ¹Pro-hGHRH(2–44)-Gly-Gly-Cys and Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys or Pro-hGHRH(1–44)-Gly-Gly-Cys, ¹Pro-hGHRH(2–44) (P < 0.05). The ¹Pro-hGHRH(2–44)-Gly-Gly-Cys showed the highest GH release from rat pituitary. The activity results showed that the N-terminal Pro modulations and the C-terminal Gly-Gly-Cys extension regulate GH release from pituitary. The results showed that the three peptides had good GH release, function-selectivity and species specificity.     

Active site of human liver aldehyde dehydrogenase

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.     

Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Interaction of Porcine Growth Hormone and Thyroxine

HAUSMAN, D.B., G.J. HAUSMAN, AND R.J. MARTIN. Endocrine regulation of fetal adipose tissue metabolism in the pig: interaction of porcine growth hormone and thyroxine. Obes Res. 1999;7:76–82. Objective : This study tested the hypothesis that combined treatment of thyroxine (T₄) and growth hormone (GH) could normalize cellular and metabolic aspects of adipose tissue development of hypophysectomized fetal pigs. Research Methods and Procedures : On day 70 of gestation, pig fetuses were hypophysectomized by microcauterization or remained intact. Hypophysectomized fetuses remained untreated or were treated from day 90 to day 105 of gestation with T₄, GH, or a combination of both hormones. Results : Body weights were unaffected by hypophysectomy or hormone treatment. De novo lipogenesis in subcutaneous adipose tissue was increased 10-fold by hypophysectomy, consistent with our previous results. This increase was abolished by GH treatment in the hypophysectomized fetuses. In contrast, T₄ treatment of the hypophysectomized fetuses resulted in a 12-fold further increase in adipose tissue lipogenesis, an effect that was negated by concomitant administration of GH. Lipolytic response to isoproterenol was decreased by hypophysectomy, unaffected by GH treatment, and restored to intact values by T₄ or by T₄+GH treatment in the hypophysectomized fetuses. Discussion : In contrast to T₄, GH does not influence serum insulin-like growth factor-I or adipose tissue lipolysis, but decreases lipogenesis in the fetal pig. However, replacing both T₄ and GH normalized hypophysectomized fetuses to a greater extent than either GH or T₄ alone. Thus, any influence of thyroid hormones on stimulating adipose tissue lipogenesis in the developing fetal pig may be normally counterregulated by pituitary-derived growth hormone.     

MAP dendrimer elicits antibodies for detecting rat and mouse GH‐binding proteins

The membrane‐bound rat GH‐R and an alternatively spliced isoform, the soluble rat GH‐BP, are comprised of identical N‐terminal GH‐binding domains; however, their C‐terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C‐terminal peptide sequence of the rat GH‐BP (GH‐BP_263–279) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP_263–279 segments onto a tetravalent Lys₂‐Lys‐β‐Ala‐OH core peptide was carried out using Fmoc chemistry. The mass of the RP‐HPLC‐purified synthetic product, 8398 Da, determined by ESI‐MS, was identical to expected mass. Three anti‐rat GH‐BP_263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10^?3, recognized both the rat GH‐BP_263–279 MAP and recombinant mouse GH‐BP with ED₅₀s within a range of 5–10 fmol, but did not cross‐react with BSA in dot blot analyses. BETO‐8041 antisera (10^?3 dilution) recognized GH‐BPs of rat serum and liver having M_rs ranging from 35 to 130 kDa, but did not recognize full‐length rat GH‐Rs. The antisera also detected recombinant mouse GH‐BPs. In summary, the tetravalent rat GH‐BP_263–279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C‐termini of both rat and mouse GH‐BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH‐BPs. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Implication of growth hormone in experimental induction of vitellogenesis by estradiol-17 beta in female silver eel (Anguilla anguilla L.)]

The effect of different preparations of growth hormone (GH) was assayed with 17 beta-estradiol on vitellogenesis in hypophysectomized or normal female silver eels. Vitellogenin (Vg) plasma levels were taken as the index of hepatic vitellogenesis. The E2 doses were chosen to give the same pattern for the plasma Vg level as in the controls. They decreased or remained undetectable in hypophysectomized or normal animals. GH also failed to induce alone a significant modification. When E2 was injected together with a GH, plasma Vg levels were 5.13 +/- 1.30 times higher with salmon GH in hypophysectomized eels and 2.01 +/- 0.25 times higher with bovine GH in normal eels. GH is shown to enhance the effects of E2 on hepatic vitellogenesis induction in a teleost.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the major protein components of the virion: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently removed HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA₂ 185–211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a major site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM β-mercaptoethanol than in the presence of 50 mM. However, the complete removal of ectodomains of HA by the high-and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein M1. The action of trans-epoxysuccinyl-L-leucylamido)(4-guanidino)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl₂ On the hydrolysis of proteins HA and M1 by bromelain was investigated.     

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1–A6), which were confirmed by Western blotting and MALDI–TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The ¹⁹⁹Val–Ser–Tyr–Asn²⁰² sequence in peptide T12 of A1 and A2 was replaced by ¹⁹⁹Leu–Ser–His–Glu²⁰² in A3 and A4 (T12′) while in A5 and A6 this sequence was ¹⁹⁹Val–Ser–Tyr–Val²⁰² (T12″). Peptide T1 showed the presence of ¹⁰Asn in the isoforms A1–A5 while in A6 this amino acid was replaced by ¹⁰Lys (T1′). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.     

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: II. Identification of a critical cysteine-containing tryptic peptide derived from the thiocyano enzyme

The pig heart TPN-speciftc isocitrate dehydrogenase was reacted successively with 5,5′-dithiobis(2-nitrobenzoic acid) and K¹⁴CN in the presence of various combinations of substrates and/or inhibitors to produce thiocyano derivatives of the reactive cysteine residues. The thiocyano-enzyme preparations, after cyclization and cleavage to yield iminothiazolidine derivatives, were digested with trypsin and the resultant tryptic digests were fractionated by two-dimensional paper chromatography and high-voltage electrophoresis. The ¹⁴C-labeled peptides were located by a two-dimensional radioscanner. The distribution of radioactivity in the tryptic digests of inactive enzyme, obtained by reaction with 5,5′-dithiobis(2-nitrobenzoate) and K¹⁴CN in the absence of ligands, was compared with that of enzyme obtained by reaction in the presence of ligands such as Mn²⁺ and isocitrate or Ca²⁺ and isocitrate which protect against inactivation. The results suggest that modification of one cysteine-containing peptide, termed Peptide 2, leads to inactivation. Peptide 2 has been purified and its amino acid composition determined. Evidence from this and the preceding paper indicates that this peptide participates in the metal-isocitrate binding site. The amino acid composition of the intact pig heart TPN-specific isocitrate dehydrogenase is also presented.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.