Short Analogues Research Articles

An improved rapid method for selecting monoclonal antibodies with high catalytic activities.

Monoclonal antibodies were raised against a beta-naphthyl phosphonate hapten (1) to elicit antibodies capable of catalyzing the hydrolysis of beta-naphthyl acetate (3). After cell fusion, potential catalytic antibody-producing hybridomas were selected, by use of a competitive inhibition assay on the basis of the binding activity for a short transition-state analogue (inhibitor 5), followed by use of high-performance liquid chromatography analysis for the hybridoma supernatants to screen the antibodies processing catalytic activities. It was shown that supernatants of 12 wells had high binding activity with inhibitor and of them, 7 had catalytic activities. After cloning by limiting dilution, we got two hybridoma clones producing monoclonal antibodies which catalyzed the hydrolysis of beta-naphthyl acetate. This combination of competitive inhibition assay with high-performance liquid chromatography analysis represents an improved rapid approach for the screening of potential catalytic antibodies and significantly increases the possibility of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (Kcat/K(uncat) approximately 10(6) and specificity.

Concise synthesis of L-α-phosphatidyl-D- myo-inositol 3,4-bisphosphate, an intracellular second messenger

A highly efficient, asymmetric total synthesis of the title phospholipid as well as short chain diester and cross-linkable diether analogs was achieved in six steps from the readily available cyclitol 1.

Identification and pharmacological characterization of somatostatin receptors in rat lung.

1. [125I]-[LTT]SRIF-28 and [125I]-SMS 201-995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]-[LTT]SRIF-28 without any significant specific binding of the sst2/sst5-receptor selective ligand [125I]-SMS 201-995. 2. In radioligand binding studies, specific binding of [125I]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]-SMS 201-995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube. 3. [125I]-[LTT]SRIF-28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8 +/- 0.1 h-1 (n = 3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7 x 10(10) M-1 h-1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k-1) = 0.44 +/- 0.07 h-1 corresponding to a half-time of 95 +/- 15 min (n = 3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]-[LTT]SRIF-28 was calculated to be 11.9 pM. 4. Saturation binding of [125I]-[LTT]SRIF-28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD = 10.3 +/- 0.1; n = 3) and a receptor density (Bmax) of 78 +/- 3 fmol mg-1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5. Specific binding of [125I]-[LTT]SRIF-28 to rat lung membranes was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 >> RC 160 > BIM 23014 > SMS 201- 995 > BIM 23056 > MK 678. 6. The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst, (r = 0.92) and sst4 (r = 0.95) receptors, respectively. 7. Reverse transcriptase--polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression. 8. The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.

R-073. Impact of long and short GnRH analogue protocols on oocyte and embryo quality

R-073. Impact of long and short GnRH analogue protocols on oocyte and embryo quality

Expression of a Non-MDR2-Coded Liver Phosphatidylcholine Membrane Transport Protein inXenopus laevisOocytes

Phosphatidylcholines (PC) are secreted into the bile via a membrane transport protein(s). Recently, evidence for ATP-dependentmdr2-encoded PC transport as well as for carrier-mediated PC transport had been reported. Therefore, we investigated whether mdr2 P-glycoprotein is involved in the transport of a water-soluble short chain phosphatidylcholine analogue L-α-dibutyroyl-PC (diC4PC) induced by expression of liver mRNA inXenopus laevisoocytes. Expression of mouse and ratmdr2cRNA did not result in diC4PC net uptake inXenopus laevisoocytes. By contrast oocytes showed a similar carrier-mediated uptake activity for diC4PC after injection of mouse, rat and human liver total mRNA (Km 7.7, 9.6, and 11.6 mM). Antisense inhibition ofmdr2mRNA expression increased diC4PC uptake induced by total liver mRNA from mouse and rat. The present data prove the existence of a specific mRNA for a non-mdr2-coded cell membrane PC carrier in mouse, rat, and human liver which exhibits similar transport affinity for diC4PC as the PC carrier in rat liver canalicular membranes.

In vitro fertilisation. A review of drug therapy and clinical management.

Since the first in vitro fertilisation (IVF) pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couple. Pregnancy rates with IVF improve as the number of high quality embryos available for transfer increases; therefore, ovarian stimulation agents to produce multiple oocysts for IVF are advantageous. Clomifene (clomiphene citrate), human menopausal gonadotrophin (hMG; menotropins), and subsequent generations of products are commonly used as stimulation agents. In conjunction with the stimulation agents, gonadotrophin-releasing hormone (GnRH) agonists and human chorionic gonadotrophin (hCG) serve as adjuvants for successful control of all events in the induction process. Clomifene, an estrogen agonists/antagonist, occupies the estrogen receptor for a longer period of time than estrogen (weeks versus hours). Because this signal is interpreted as low estrogen, GnRH is released, which produces a rise in circulating levels of follicle-stimulating hormone (FSH) and luteinising hormone (LH) and subsequent ovarian follicular development. Menotropins is collected by passing urine from menopausal donors over a Sepharose column, followed by removal of high molecular weight impurities by chromatography. The mixture of FSH and LH is biologically standardised. This product stimulates multiple ovarian follicular development. Urofollitrophin is produced using antibodies to hCG anchored to a separation column. LH then can be excluded from the eluate by binding to the hCG antibodies (LH immunoaffinity column). Highly purified FSH is obtained by passing menopausal urine over a column with monoclonal antibodies to FSH. The isolated FSH is then eluted from the column by a highly basic solution and crystallised. This product delivers FSH at a 90% purity and can be administered subcutaneously rather than intramuscularly. Dosage is standardised on a mg/kg basis. Recombinant human FSH is completely free of LH and offers the advantages of better batch consistency, greater purity, and absence of any human contaminants. It may be given both subcutaneously and intravenously. Genetically engineered FSH combines portions of the native protein with another protein (hCG) which enhances its potency and extends the half-life compared with wild-type FSH. Short, medium and ultra-long activity analogues of genetically engineered FSH may be used to tailor stimulation protocols in various clinical situations. Growth hormone is an adjuvant to ovarian stimulation which results in a decreased number of ampoules of menotropins being required to achieve ovulation in poor responders. Ovulation triggers include both hCG and GnRH agonists. Progesterone supplementation is generally used in the luteal phase of the IVF cycle and is administered by intramuscular injection or vaginal suppository. It appears that conscious sedation with midazolam, pethidine (meperidine) and fentanyl is nontoxic for oocyte recovery. If full anaesthesia is required for gamete intrafallopian tube transfer (GIFT) or zygote intrafallopian tube transfer (ZIFT), balanced anaesthesia with nitrous oxide and an opioid appears to be the most appealing option. Appropriate information on the clinical use of the drugs used in IVF greatly reduces patient stress associated with the complex multidrug regimens associated with the procedure.

Binding properties of somatostatin receptor subtypes

In the past few years, five different somatostatin (SRIF) receptor subtypes (sst 1–5) have been identified, which form a distinct group in the superfamily of G-protein—coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst 1–5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201–995, RC 160, or BIM 23014, primarily interact with the sst 2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst 2 (MK 678), the sst 3 (BIM 23056), or the sst 5 (BIM 23052, L362–L855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst 1–5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst 2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst 1 for SMS 201–995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst 1, almost full sst 2-like binding of SMS 201–995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pK i values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst 4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst 4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [ 125I]Tyr 3-SMS 201–995 was detected, since SMS 201–995 has low affinity for sst 4. In contrast, specific binding of [ 125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst 4 receptors.

Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans.

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.

Platelet-activating factor and its structural analogues in the earthworm Eisenia foetida

The earthworm Eisenia foetida was shown to contain large amounts of ether-containing phospholipids such as alkylacylglycerophosphocholine (61.3% of choline glycerophospholipids) and alkenylacylglycerophosphoethanolamine (66.0% of ethanolamine glycerophospholipids). We also found a substantial amount of ether-containing PAF-like lipid in this animal, its level being increased after the animal is injured. We showed evidence that this PAF-like lipid consists of PAF and PAF analogues containing short chain fatty acids other than acetic acid. Notably, a propionic acid-containing species but not PAF itself, is the most predominant species in this animal. We also confirmed that the earthworms contain enzyme activities involved in the synthesis of PAF and short chain fatty acid-containing PAF analogues. Interestingly, the acetyltransferase activity in earthworms is resistant to high concentrations of the substrate lysophospholipid. Thus, both the structure of the PAF-like lipid and the properties of the enzymes involved in PAF-like lipid metabolism in the earthworms are somewhat different from those in mammalian tissues.

Involvement of septide-sensitive tachykinin receptors in inositol phospholipid hydrolysis in the rat urinary bladder.

The selective NK2 agonist [Lys5-MeLeu9,Nle10]NKA(4-10) markedly stimulated [3H]inositol monophosphate (PI1) formation in prisms from the rat urinary bladder. This response was blocked by the NK2 antagonist SR 48968. Senktide (NK3 agonist) was inactive. Septide, a short SP analogue, and the NK1 agonists [Pro9]SP and [Sar9,Met(O2)11]SP also stimulated [3H]IP1 formation and several NK1 tachykinin antagonists (RP 67580, CP 96345, GR 82334, and [D-Pro9,t beta-BPr10,Trp11]SP) were more potent in blocking the septide than the [Pro9]SP response. GR 82334 was the most discriminative. SR 48968 (10(-6) M shifted the [Pro9]SP dose-response curve but did not modify the septide dose-response curve. Septide had a low affinity for [3H][Pro9]SP binding sites, suggesting further that septide and NK1 agonists act on different receptors. Finally, both [Pro9]SP and [Sar9,Met(O2)11]SP blocked the septide-evoked response, acting as partial agonists at the septide-sensitive tachykinin receptors.

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms.

The physiological significance of the occurrence of sequence similar antimicrobial peptides in frog skin, as the bombinins in Bombina, the magainins in Xenopus, and the dermaseptins in Phyllomedusa, is a major unanswered question. Dermaseptins s1, s2, s3, s4, and s5, a family of cationic (lysine-rich), amphipathic antifungal peptides of 28-34 residues were thus synthesized, purified to homogeneity, and evaluated for their growth-inhibition activity in vitro against various pathogenic microorganisms. Although all five of these peptides shared a similar spectrum of lytic activity against the filamentous fungi that are responsible for opportunistic lethal infections that follow the immunodeficiency syndrome or the use of immunosuppressive agents, they exhibited marked differences in their potencies to arrest the growth of Gram-positive and Gram-negative pathogenic bacteria and yeasts. Likewise, whereas dermaseptins s1 and s5 were devoid of hemolytic activity, dermaseptin s4 caused lysis of erythrocytes at micromolar concentrations. The dermaseptins exhibited dramatic synergy of action upon combination, resulting in some cases in a 100-fold increase in antibiotic activity of the mixture over the activity of the peptides separately. Shortening the peptide chain of dermaseptin s3 to dermaseptin s3-(1-16)-NH2 did not affect the antimicrobial potency of the peptide. Further reduction of the chain length yielded peptide derivatives gradually showing reduced activity. Surprisingly, however, analogs of dermaseptin s3 as shorter as 10-12 residues in length remained fully active against Enterococcus faecalis, Cryptococcus neoformans, and against Aeromonas caviae, the causal agent of red-leg disease in amphibians. Overall, these results suggest that, despite 40% sequence similarities, the dermaseptins have distinct spectra of anti-microbial activity and may act in concert to circumvent host invasion by providing frogs with a better shielding against a broad array of microorganisms. They also demonstrate the potential usefulness of short analogs of these peptides as potential candidates for biorational design of germicides.

Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.

Permeabilization of the mitochondrial inner membrane by short cecropin-A-melittin hybrid peptides.

A number of cecropin-A-melittin hybrid peptides have previously been shown to be potent antibacterial agents [Andreu, D., Ubach, J., Boman, A., Wahlin, B., Wade, D., Merrifield, R. B. & Boman, H. G. (1992) FEBS Lett. 296, 190-194]. In the present report we analyze their action on biological systems using rat liver mitochondria as a test system. We demonstrate that the longest peptide, cecropin-A-(1-8)-melittin(1-18) permeabilizes the mitochondrial inner membrane allowing the movement of both charged and non-charged solutes. Concentrations used have already been shown to be bactericidal. This effect is also demonstrated under respiring conditions where succinate oxidation is uncoupled. Shorter analogs also permeabilize mitochondria although at ten-fold higher concentrations. Heparin potentiates the peptide effects at low concentrations, while at high concentration it becomes inhibitory. We propose that the cecropin-melittin analogs disrupt the mitochondrial membrane in a detergent-like mode rather than by creating selective channels as had been previously suggested.

Inhibition of parathyroid hormone secretion by amino-terminal chromogranin peptides.

The parathyroid gland responds to decreases in levels of extracellular calcium by increasing the secretion of both PTH and chromogranin-A (CGA) in approximately equal molar ratios. Because CGA has been suggested to be a precursor for biologically active peptides, we used primary cultures of bovine parathyroid cells to examine the effects of various peptides from CGA as well as analogous peptides from chromogranin-B (CGB) on PTH secretion. In concentrations from 10(-8)-10(-7) M, amino-terminal peptide CGA-(1-76) effectively inhibited the release of PTH in response to low extracellular calcium. Truncated analogs of this peptide, CGA-(1-40), CGB-(1-41), and CGA-(17-38) were also found to be active in the following order: CGA-(1-76) = CGA-(1-40) = CGB-(1-41) > CGA-(17-38). The biological activity of CGA-(1-40) was markedly reduced after reduction and alkylation, which resulted in disruption of the single disulfide bond between Cys17 and Cys38. Moreover, peptides derived from other regions of CGA and CGB, which included CGA-(403-428), CGB-(1-16), CGB-(316-326), and CGB-(635-657) were inactive. Pulse-chase experiments, using primary cultures of bovine parathyroid cells, revealed the presence of a CGA peptide in the culture medium that had the same amino-terminal sequence and mobility on sodium dodecyl sulfate-polyacrylamide gels as synthetic CGA-(1-76). Furthermore, in binding and cross-linking studies using intact parathyroid cells, CGA-(1-40) formed a single, covalently linked protein complex with a mol wt of 78,000. Formation of the protein complex could be completely inhibited in the presence of an excess of either CGB-(1-41) or CGA-(17-38). These results show that a naturally occurring amino-terminal peptide from CGA as well as shorter analogs can act as potent inhibitors of PTH secretion, and that their biological activity may be mediated through binding to a specific cell surface protein.

Synthesis of an edothelin cyclic analogue using an original multidimensional protection scheme

A short cyclic analogue of endothelin 1 has been synthesized. The native disulphide bridges between Cys 3-Cys 11 and Cys 1-Cys 15 have been replaced respectively by two β Ala and a diaminopropionic acid linked to an aspartic acid.

Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

Evidence for the presence of a phosphatidylcholine translocator in isolated rat liver canalicular plasma membrane vesicles.

In the present study we used the water-soluble short chain phosphatidylcholine analogue L-alpha-dibutyryl-glycero-3-phosphatidylcholine (diC4PC) to investigate the mechanism involved in the canalicular secretion of phospholipids in rat liver. Uptake of 14C-labeled di-C4PC was studied in isolated microsomes as well as in basolateral (sinusoidal) and canalicular plasma membrane vesicles. Saturable uptake of diC4PC into an osmotically active space was observed in microsomes and canalicular membrane vesicles. In contrast, diC4PC uptake into basolateral membrane vesicles could be accounted for by cross-contamination with endoplasmic reticulum and canalicular membrane vesicles. Whereas the Km values for diC4PC uptake (37 degrees C) were similar in microsomes (7.4 +/- 2.6 mM) and canalicular membrane vesicles (8.2 +/- 2.0 mM), the Vmax values were approximately 2-fold higher in canalicular membrane vesicles (29.6 +/- 2.7 nmol/mg of protein x min) than in microsomes (16.7 +/- 2.1 nmol/mg of protein x min). Furthermore, Pronase treatment of the membrane vesicles reduced diC4PC uptake by 34-54% in both subfractions, whereas the D-[14C]glucose-accessible water space was only reduced by approximately 20%. These data provide direct evidence for the presence of a protein-mediated phosphatidylcholine translocating activity in the canalicular membrane of rat hepatocytes. This canalicular "flippase" has kinetic properties similar to those described previously in microsomes and provides a potential pathway for the translocation of bile salt dissolvable biliary phospholipids to the exoplasmic leaflet of the canalicular membrane.

Membrane-modifying properties of the pore-forming peptaibols saturnisporin SA IV and harzianin HA V.

Harzianin HA V and saturnisporin SA IV are alpha-amino isobutyric-containing peptides with 18- and 20-residue chain length, respectively. They were isolated from in vitro cultures of Trichoderma species and their sequences were determined by the combined use of positive ion FAB mass spectrometry and NMR. In organic solvent solution, both peptides exhibited the same predominant alpha-helical secondary structure including a hinge at the level of the central Pro residue, as deduced from NMR data. Their interaction with neutral phospholipid bilayers was shown to induce leakage of the material entrapped in small unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3). When incorporated into neutral planar lipid bilayers, they promoted voltage-gated channels. The concentration- and voltage-dependences of the ionic conductances induced by these peptides were studied in macroscopic current-voltage experiments. Single-channel measurements showed that whilst SA IV developed non-integral multi-open states similar to those induced by alamethicins, but with faster kinetics, the shorter analogue, HA V promoted much smaller-sized conducting aggregates in agreement with macroscopic conductance data.

Substrate based inhibitors of smooth muscle myosin light chain kinase

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.