Carboxyl-terminal Peptide Research Articles

The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa.

A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl. The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea. Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity. The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta. Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214. The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin. The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein.

The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.

Proteolytic release of human angiotensin-converting enzyme. Localization of the cleavage site.

Angiotensin-converting enzyme (EC 3.4.15.1, ACE) is a transmembrane protein with a short carboxyl-terminal cytoplasmic domain, a 17-amino acid hydrophobic anchor domain, and a large N-terminal extracellular region containing two catalytically homologous domains. An active soluble form of ACE circulates in human plasma and is produced in culture medium of Chinese hamster ovary (CHO) cells transfected with the full-length human ACE cDNA. The mechanism of ACE release in CHO cells involves a post-translational proteolytic cleavage occurring in the carboxyl-terminal region. The carboxyl terminus of the secreted recombinant ACE, AGQR, was established by carboxyl-terminal microsequencing and corresponds to a cleavage site between Arg-1137 and Leu-1138. Two independent studies confirmed this proposed cleavage site: amino acid analysis of a carboxyl-terminal peptide derived from soluble ACE and immunocharacterization of membrane-bound and soluble ACE with antibodies raised against three peptides located along the carboxyl-terminal ACE sequence. In order to assess the importance of Arg-1137, this amino acid was mutated to a glutamine residue. This mutation did not prevent the secretion of ACE, suggesting that the solubilizing enzyme can accommodate this change or can use an alternative cleavage site. Finally, the production of soluble ACE in CHO cells appears to be proportional to the level of cellular ACE, implying that the solubilizing enzyme is not a limiting factor. In addition, the carboxyl-terminal sequence of the human plasma ACE was identified as AGQR, thus supporting the fact that a similar mechanism could operate in human vascular cells.

Structural basis of pilus subunit recognition by the PapD chaperone.

The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.

Specific immunostaining for CCP II, a novel calcitonin carboxyl terminal peptide encoded by the calcitonin/CGRP gene.

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way.

Determination of Carboxyl-Terminal Residue and Bisulfide Bonds of MACIF (CD59), a Glycosyl-Phosphatidylinositol-Anchored Membrane Protein1

MACIF (CD59) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane glycoprotein which inhibits the formation of membrane attack complex of human complement. MACIF prepared from human erythrocyte membranes was digested with pronase. When the digest was subjected to two-phase partition with butanol and 0.1 N HCl, the carboxyl-terminal peptide was recovered in the butanol phase because of the attachment of the highly hydrophobic GPI. The amino acid sequence of the peptide was determined to be Asn72 at its amino-terminus and up to Glu76, while the presence of Asn77 was ambiguous. To allow unequivocal determination of the carboxyl-terminus, a soluble form of MACIF was prepared from human urine on a large scale. The carboxyl-terminal peptide from the soluble form was prepared by tryptic digestion followed by reversed-phase HPLC. The sequence and composition of the peptide unequivocally revealed Asn77 as the carboxyl-terminus. The pattern of disulfide bonds of MACIF was also determined with the membrane form as well as the soluble form. Cystine-containing peptides were prepared by chymotryptic and tryptic digestion, purified by HPLC, and their amino acid sequences were determined. The results indicated that disulfide bonds were formed at Cys3-Cys26, Cys6-Cys13, Cys19-Cys39, Cys45-Cys63 (or 64), and Cys63 (or 64)-Cys69.

A wheat (Triticum aestivum) cDNA clone encoding a plastid-localized heat-shock protein.

Higher plants produce both high mo1 wt (in the range of 70,000-110,000) and LMW (in the range of 15,000-30,000) HSPs in response to heat-stress conditions. During periods of heat stress, plant cells produce an abundance of LMW HSPs that are encoded by gene families (Vierling, 1991). The physiological function of these proteins remains obscure, but the evolutionary conservation of this response in higher plants suggests that they perform some important function in protection of plants from heat. LMW HSP genes have been isolated from a variety of crop plants such as soybean, pea, carrot, wheat (Triticum aestivum), and corn. Recently, Vierling (1991) classified these proteins into four families, two of these encode cytoplasmic proteins, one represents endomembrane proteins, and one includes the plastid-localized HSP genes. The question of nonfortuitous association of LMW HSPs with plastids during and after heat shock has been addressed by severa1 research groups who concurred that HSPs with mo1 wts ranging from 21,000 to 28,000 are specifically localized in chloroplasts during and after heat stress (for review, see Vierling, 1991). These nuclear-encoded HSPs consist of an amino-terminal transit peptide region and a carboxylterminal mature peptide that has significant similarity with other cytoplasmic LMW HSPs. The translation products of these mRNAs are transported to plastids where they are associated with either the soluble stroma portion or the grana region of the thylakoid membranes. Comparison of the amino acid sequences of plastid-localized HSPs from petunia, Arabidopsis, pea, soybean, and corn has led to identification of three conserved regions designated I, 11, and 111 (Chen and Vierling, 1991). Regions I and I1 are also shared by cytoplasmic HSPs, but region 111 is exclusively present in plastidlocalized HSPs. The first monocotyledonous cDNA encoding a plastidlocalized HSP26 was isolated from corn by Nieto-Sotelo et al. (1990). Using this cDNA clone as a probe, we previously isolated a cDNA clone from wheat, which encodes an HSP of mo1 wt of 26,600 (Tahsp26.6a) (Weng et al., 1991). We

Synthesis of Barnase Site-Specifically Labelled with Two 13C Atoms Using Partially Protected Peptide Thioester Building Blocks

Abstract Barnase, site-specifically labelled with two 13C atoms, was synthesized using partially protected peptide thioesters as building blocks. Four partially protected peptide segments (Boc–[Lys(Boc)19,27]–barnase(1—34)–SC(CH3)2CH2CO–Nle–NH2, iNoc–[Lys(Boc)39,49]–barnase(35—52)–SC(CH3)2CH2CO–Nle–NH2, iNoc–[Lys(Boc)62,66]–barnase(53—81)–SC(CH3)2CH2CO–Nle–NH2, [Lys(Boc)98,108]–barnase(82—110)) were successively condensed in the presence of silver ions and N-hydroxysuccinimide (HONSu). Finally, barnase with full ribonuclease activity was obtained in a yield of 11% based on the carboxyl terminal peptide segment.

Structure of the alpha subunit of F1-ATPase probed by limited proteolysis.

The structure of the isolated alpha subunit of F1-ATPase from the thermophilic Bacillus strain PS3 was probed using limited proteolysis by four different proteases, and the following results were obtained. 1) Distribution of 21 protease-cleaved sites is similar to that of the beta subunit of F1-ATPase (Tozawa, K., Odaka, M., Date, T., and Yoshida, M. (1992) J. Biol. Chem. 267, 16484-16490), thus providing experimental evidence for similar folding topology of the two subunits, and the locations of 11 water-exposed loop regions in the tertiary structure are predicted. 2) Most proteolytic peptides remain associated to maintain the gross structure of the alpha subunit and can reassociate each other after denaturing urea treatment. 3) However, the carboxyl-terminal peptides comprising approximately 80 residues (C1 peptides) are released from other peptide(s) during proteolysis, and those comprising approximately 105 residues (C2 peptides) are released during native polyacrylamide gel electrophoresis after proteolysis. 4) Inclusion of Mg-ATP in the native electrophoretic system prevents the release of the C2 peptide. Addition of Mg-ATP to the proteolysis mixtures results in an increase of the C2 peptide population and a decrease of the C1 peptide population. Thus, Mg-ATP induces a conformational change at the regions of C1 and C2 peptides of the alpha subunit. 5) Except for the trypsin-treated one, protease-treated alpha subunits are reconstitutable with the native beta subunit into the form of alpha 3 beta 3 complexes, which show significantly higher ATPase activities than the intact alpha 3 beta 3 complex. This activation is attributable to the cleavage of a peptide bond that produces C2 peptides. The carboxyl-terminal region of the alpha subunit is likely to be involved in the regulation of ATPase activity in F1-ATPase.

Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography

The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by “chymotryptic-like” cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.

Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67-94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)73,87] 67-94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.

Biochemical and functional characteristics of soluble MHC molecules derived from H-2Ld/Q10d chimeric gene.

We have constructed a chimeric class I gene in which the 5' half of the H-2Ld gene is linked to the 3' half of Q10d. The resulting H-2Ld/Q10d protein is homologous to the native H-2Ld heavy chain for the three external domains except for an Arg to His substitution at position 260. The transmembrane and intracytoplasmic domains of the H-2Ld chain are replaced by the short low hydrophobic transmembrane-like domain of the Q10d chain. Following DNA-mediated gene transfer into mouse L cells, transformants were selected for the presence of specific mRNA. Radiolabelling and immunoprecipitation analysis revealed secretion of a 48-46 kd chain weakly associated with beta 2-microglobulin. This molecule reacts with H-2Ld-specific mAb that identify determinants on the first and second domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum, but is not recognized by a mAb specific for a determinant of H-2Ld third domain. The integrity of antibody reactivity of the first and second domains together with beta 2-microglobulin association suggest that our molecule may be considered a good soluble counterpart of the native membrane H-2Ld molecule with which to perform functional studies. In order to analyze the immunogenic capacities and T-cell recognition of the soluble H-2Ld molecules, T-cell lines were produced from mice of various inbred strains immunized with supernatant from H-2Ld/Q10d-transfected fibroblasts. Characterization of these T cells revealed that they expressed a CD4+CD8- phenotype, and recognized H-2Ld/Q10d products in a class II-restricted manner.

Epitope mapping of histone 5 (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera

To define the linear epitopes on H5 that react with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) sera, concurrent overlapping hexameric peptides corresponding to the sequence of H5 were synthesized by stepwise elongation of the polypeptide chains on polyethylene supports. The hexapeptides were tested for reactivity with 8 SLE and 8 DIL sera using an enzyme linked immunosorbent assay (ELISA). SLE and hydralazine-induced lupus (HIL) antibodies were most reactive with peptide 45 (SSRQSI) and patients with procainamide-induced lupus (PIL) were most reactive with peptide 24 (SHPTYS). The epitopes of highest reactivity were in the globular domain of H5. Low reactivity was observed with carboxyl terminal peptides. These findings differ from immunoblotting studies of protease cleaved peptides which have previously shown that the H5 determinants are in the carboxyl terminus.

Characterization of the protease and other products of amino-terminus-proximal cleavage of the herpes simplex virus 1 UL26 protein.

The herpes simplex virus 1 UL26 open reading frame encodes a protease which cleaves a small carboxyl-terminal peptide of itself and its substrate encoded by an overlapping, 3'-coterminal transcriptional unit, designated UL26.5. The translational product of UL26.5 is infected-cell protein 35c,d (ICP35c,d) (F. Liu and B. Roizman, J. Virol. 65:206-212, 1991; F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). The protease activity maps at the amino terminus of UL26 translation product designated Pra. Cleavage of Pra to remove the carboxyl-terminal 25 amino acids converts the protein to Prb (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992). Other studies reported a second, amino-terminus-proximal cleavage in UL26 gene products made in Escherichia coli (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol 66:7362-7367, 1992; C. L. DiIanni, D. A. Drier, I. C. Deckman, P.J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem., 368:2048-2051, 1993). We report the following results. (i) The amino-terminus-proximal cleavage of UL26 protein in eukaryotic cells generates two polypeptides, an apparent M(r)-25,000 amino-terminal polypeptide designated Prn and a carboxyl-terminal polypeptide which corresponds in electrophoretic mobility to ICP35a. Cleavage of the carboxyl-terminal 25 amino acids by the UL26 protease converted ICP35a to ICP35b. (ii) Replacement of Ala-247-Ser-248 with Arg-Pro precluded the amino-terminus-proximal cleavage. (iii) Prn, the amino-terminal product of the cleavage reaction at amino acid 247 functions as a protease. (iv) Additional amino acid substitutions in the putative domain of the protease yielded results consistent with the hypothesis that UL26 encodes a serine protease. (v) The domain of the UL26 protein whose modification confers the formation of double bands for all products (Pra, Prb, ICP35a, ICP35b, ICP35c,d, and ICP35e,f) except Prn maps in the domain shared by UL26 and UL26.5, between codons 307 and 417.

A Method for Identifying the Carboxy Terminal Amino Acid of a Protein

A highly sensitive new method for identifying the carboxy terminus of a protein was developed. The carboxyl terminal amino acid was racemized by reaction with acetic anhydride. The resulting modified protein was subjected to acid hydrolysis. The hydrolysate was denyatized with (+)-1-(9-fluorenyl)ethyl chloroformate to give fluorescent amino acid diastereomers. The amino acid diastereomers were separated on a reversed-phase column. Only carboxyl terminal amino acids give a D-amino acid. Application of this method was described for the isolation and identification of carboxyl terminal peptides from an enzymatic digest of a protein.

Distribution of proneuropeptide Y-derived peptides in the brain of Rana esculenta and Xenopus laevis.

The distribution of proneuropeptide Y-containing perikarya and nerve fibers in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward neuropeptide Y and the carboxyl terminal flanking peptide. The distribution of proneuropeptide Y-like immunoreactivity was similar in both anurans. In the telencephalon, immunoreactive perikarya were found in the olfactory bulb, all subdivisions of the pallium, the septum, pars lateralis of the amygdala, the nucleus accumbens, and the anterior preoptic area. In the diencephalon, labelled perikarya were detected in the ventromedial, ventrolateral and central thalamic nuclei, the magnocellular preoptic nucleus, the suprachiasmatic nucleus, the posterior tuberculum, and the infundibulum. Amacrine-like cells were stained in the retina. In the pretectal area, posterior thalamic neurons showed intense, Golgi-like immunostaining. In the mesencephalon, immunoreactive cells were found in the reticular nucleus, the anteroventral tegmental nucleus, the optic tectum, the interpeduncular nucleus, and the torus semicircularis. In the rhombencephalon, labelled perikarya were detected in the secondary visceral nucleus, the central gray, the nucleus of the solitary tract, the dorsal column nuclei, and the spinal nucleus of the trigeminal nerve. Immunoreactive nerve fibers were observed in all areas of the brain that contained labelled perikarya. The densest accumulations were found in the accessory olfactory bulb, pars lateralis of the amygdala, the ventral habenula, the posterior pituitary, the optic tectum, the interpeduncular nucleus, and the saccular nucleus. The distribution of proneuropeptide Y-like immunoreactivity in the anuran brain showed many similarities to the distribution described for the amniote brain.

Procollagen Type-I in the Serum and Dialysate of Continuous Ambulatory Peritoneal Dialysis Patients

Procollagen-I carboxylterminal extension peptide (PICP) was determined in the serum and dialysate of 26 continuous ambulatory peritoneal dialysis (CAPD) patients and in the serum of 11 healthy controls. PICP serum levels were significantly higher in CAPD patients than in healthy controls (p < 0.001). There was no correlation between serum PICP levels and those of calcium, phosphorus, magnesium, alkaline phosphatase, osteocalcin, and intact parathyroid hormone (iPTH). Serum and dialysate levels of osteocalcin and iPTH showed a significant correlation (p < 0.001). The dialysate-to-serum PICP ratio in 21 patients was lower than 1.0. In the remaining 5 patients, however, the above ratio was higher than 1.0. We conclude that in CAPD patients serum PICP levels do not correlate with biochemical parameters of renal osteodystrophy. A dialysate-to-serum PICP ratio above 1.0 could implicate an increased local peritoneal fibroblastic activity and could be a useful marker of peritoneal fibrosis in CAPD.

Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides.

The induction of granulosa cell differentiation and follicle maturation is dependent upon the stimulatory actions of FSH. Our recent studies used recombinant DNA technology to fuse the carboxyl-terminal peptide (CTP) of hCG beta-subunit to the carboxyl-terminus of the FSH beta-subunit. The resulting FSH analog has identical in vitro receptor-binding and biological activities as wild-type FSH (WT-FSH), but an increased circulating half-life. The present studies examined further the ability of FSH with one (FSH-CTP1) or two (FSH-CTP2) appended CTPs to promote granulosa cell differentiation and follicle ovulatory potential. WT-FSH, FSH-CTP1, and FSH-CTP2 were produced from Chinese hamster ovary cells transfected with the common alpha-subunit and respective beta-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTP1 and FSH-CTP2 retained full FSH receptor-binding activity, but did not bind LH receptors. To compare in vivo bioactivity, immature estrogen-primed female rats received ip injections of FSH or the agonists at 0 and 24 h. At 48 h, substantial stimulation (up to 2.5-fold) of ovarian weight was induced by 1.0 and 3.0 IU/day FSH-CTP1 or FSH-CTP2, whereas a higher dose (10 IU/day) of WT-FSH was required for an 1.8-fold stimulation. Although the in vivo potencies of FSH-CTP1 and FSH-CTP2 were similar, FSH-CTPs were about 10-fold more potent than WT-FSH in inducing granulosa cell aromatase activity and LH receptors. We further reduced the frequency of hormone administration. Increasing doses (1-10 IU) of a single ip injection of FSH-CTP1 resulted in dose-dependent increases in granulosa cell aromatase activity and LH receptor content 48 h later. Although a single injection (10 IU) of WT-FSH had no effect, the same total dose of WT-FSH administered as four 2.5-IU injections 12 h apart was effective. To test the ovulatory potential of ovarian follicles, rats received a single injection of FSH-CTP1, followed 52 h later by 5 IU hCG to induce ovulation. Although hCG did not induce ovulation in females receiving a single dose (10 IU) of WT-FSH, 20 +/- 2 and 43 +/- 5 ovulated ova/rat were found in animals primed with 3 and 10 IU FSH-CTP1, respectively. Because twice daily injections of WT-FSH (2.5 IU/injection) also increased the ovulatory potential of the ovary, the enhanced effectiveness of FSH-CTP1 appears to be related to its increased circulating half-life.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of beta-subunit of GTP-binding regulatory protein in mitotic spindle.

The heterotrimeric GTP-binding regulatory protein (G protein) is important in membrane signal transduction. Since the function of the beta-subunit of G protein (G beta) in tumor cells is not well-documented, identification of G beta in tumor cells was performed. Immunolocalization, Western blotting, and immunoprecipitation of G beta in mammalian tumor and normal cells were investigated using rabbit antisera against amino and carboxyl terminal peptide fragments of G beta. A human nasopharyngeal carcinoma cell line (NPC-TW039) was used as the cell model because of its short doubling time. Anti-G beta immunoreactivity was found to be associated with the plasma membrane and the mitotic spindle throughout the mitotic phase of cell replication. Colcemid pretreatment resulted in random distribution of the anti-G beta reaction product in the mitotic cells. The same phenomenon was also seen in various other tumor and normal cell lines. When solubilized membranous, cytosolic, and mitotic spindle fractions were analyzed by Western blotting, G beta (35 +/- 1 kDa) was found to be associated with the plasma membrane and mitotic spindle fractions. Immunoprecipitation of isolated mitotic spindles with anti-G beta further confirmed these results. These findings indicate that G beta is closely associated with the mitotic spindle as well as the plasma membrane and may be important in regulation of cell mitosis in addition to transmembrane signal transduction.

Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides

Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides