Osteocalcin binds tightly to the gamma-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins.

Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site.

Processing, axonal transport and cardioregulatory functions of peptides derived from two related prohormones generated by alternative splicing of a single gene in identified neurons VD1 and RPD2 of Lymnaea.

The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and -B preprohormones. Here, we report the isolation and structural characterization of alpha 1, alpha 2 and beta peptides from dissected neurons VD1 and RPD2. The alpha 1 and alpha 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas beta peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the alpha 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic alpha 1 and alpha 2 peptides on isolated auricle preparations of the Lymnaea heart were examined. The two alpha peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, through non-identical, sets of peptides.

The sodium influx stimulating peptide of the pulmonate freshwater snail Lymnaea stagnalis.

In Lymnaea stagnalis integumental Na+ uptake is stimulated by the sodium influx stimulating (SIS)-peptide. Its primary structure was determined as: SRTQSRFAS- YELMGTEGTECVTTKTISQICYQCATRHEDSFVQVYQECCKKEMGLREYCEEIYTELPIRSGLWQPN++ +. Antisera raised against parts of SIS-peptide stained neurons in the visceral, parietal, and pleural ganglia, and in the proximal parts of the intestinal, anal, and right internal pallial nerves. Locations and axon projection patterns of these neurons suggest that they represent the previously described neurosecretory yellow cells.

Congenital deficiency of all vitamin K-dependent blood coagulation factors due to a defective vitamin K-dependent carboxylase in Devon Rex cats.

Two Devon Rex cats from the same litter, which had no evidence of liver disease, malabsorption of vitamin K or chronic ingestion of coumarin derivatives, were found to have plasma deficiencies of factors II, VII, IX and X. Oral treatment with vitamin K1 resulted in the normalization of these coagulation factors. After taking liver biopsies it was demonstrated that the coagulation abnormality was accompanied by a defective gamma-glutamyl-carboxylase, which had a decreased affinity for both vitamin K hydroquinone and propeptide. This observation prompted us to study in a well-defined in vitro system the possible allosteric interaction between the propeptide binding site and the vitamin K hydroquinone binding site on carboxylase. It was shown that by the binding of a propeptide-containing substrate to gamma-glutamylcarboxylase the apparent KM for vitamin K hydroquinone is decreased about 20-fold. On the basis of these in vitro data the observed defect in the Devon Rex cats can be fully explained.

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis.

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.

Primary structure and origin of schistosomin, an anti-gonadotropic neuropeptide of the pond snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata, a peptide called schistosomin is released from the central nervous system, which counteracts the bioactivity of a number of gonadotropic hormones. This leads to inhibition of the reproductive activities of the infected snail. In order to determine the structure of schistosomin, the neuropeptide was purified from the central nervous system using gel-permeation chromatography and reverse-phase h.p.l.c. The complete primary structure of the peptide was determined by N-terminal sequencing and peptide mapping. Schistosomin is a single-chain molecule of 79 amino acids with a molecular mass of 8738 Da. The peptide contains eight cysteine residues which may give rise to four intramolecular disulphide bridges that fold the peptide into a stable globular structure. A database search did not reveal any known peptides that show significant sequence similarity to schistosomin. By means of immunocytochemistry, the peptide was shown to be localized in the growth-controlling neurosecretory light green cells, which are located in the cerebral ganglia of the central nervous system of Lymnaea. In addition to schistosomin, these neurons are known to produce various insulin-related peptides.

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail.

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.

Characterization of a Gla-containing protein from calcified human atherosclerotic plaques.

In this article we describe the isolation of a 4-carboxyglutamic acid (Gla)-containing protein from calcified human atherosclerotic plaques. The protein was extracted from pulverized calcified plaques by demineralization with ethylenediaminetetraacetate and was subsequently purified by anion-exchange fast protein and high-performance liquid chromatography by using ion-exchange and gel-filtration columns. The protein was designated as plaque Gla protein (PGP) and has an apparent mass of 23 kD as estimated from sodium dodecyl sulfate-polyacrylamide gel analysis. By determining the sequence of its first six amino acid residues, the protein was unequivocally demonstrated to be not related to any other known protein. Moreover, no immunological relationship (as tested by Western blot analysis) was found between PGP and other known Gla-containing proteins.

Molluscan insulin-related neuropeptide promotes neurite outgrowth in dissociated neuronal cell cultures.

The formation of neurites in isolated neurones of the snail Lymnaea stagnalis in primary culture was studied. The insulin-related neuropeptide (MIP: Molluscan insulin-related peptide) produced by the neuroendocrine light green cells (LGCs) of Lymnaea stimulated neurite formation, both in isolated unidentified central neurons and in the LGCs. The effect of MIP was dose dependent. It was significant from the second day of culture and amounted up to an 8-fold increase in neurite outgrowth after 3 days. The results add a functional aspect to the evolutionary relationship of MIP with mammalian insulin and insulin-related peptides and suggest that the LGCs, which stimulate growth, are also involved in development of the nervous system.

Discharge induction in molluscan peptidergic cells requires a specific set of autoexcitatory neuropeptides.

The peptidergic caudodorsal cells of the pond snail Lymnaea stagnalis generate long lasting discharges of synchronous spiking activity to release their products. During caudodorsal cell discharges a peptide factor is released which induces similar discharges in silent caudodorsal cells [Ter Maat A. et al. (1988) Brain Res. 438, 77-82]. To identify this factor, the electrophysiological effects of putative caudodorsal cell gene products, calfluxin, caudodorsal cell hormone, four alpha caudodorsal cell peptides and three beta caudodorsal cell peptides, were tested individually and in various combinations. Calfluxin, alpha caudodorsal cell peptide and beta 1 caudodorsal cell peptide each had no effect on membrane potential or excitability of the caudodorsal cells. All other caudodorsal cell peptides caused excitatory responses, but did not induce discharges. Instead, only a specific combination of four caudodorsal cell peptides, caudodorsal cell hormone and alpha caudodorsal cell peptide (1-11, 3-11 and 3-10), evoked caudodorsal cell discharges with similar characteristics to electrically evoked discharges. Incomplete versions of this combination failed to cause a discharge. In addition, antibodies to caudodorsal cell hormone or alpha caudodorsal cell peptide reduced caudodorsal cell excitability and prevented the generation of discharges by electrical stimulation. These results suggest that excitatory autotransmission caused by four caudodorsal cell peptides provides a means to amplify excitatory inputs, thus leading to the generation of the all-or-nothing caudodorsal cell discharge.

Structure of one of the neuropeptides of the egg-laying hormone precursor of Lymnaea.

The neurosecretory caudo-dorsal of the freshwater pulmonate snail Lymnaea stagnalis, a peptidergic system controlling egg laying and egg-laying behavior, produce several neuropeptides. One of these peptides, calfluxin, causes the influx of Ca2+ into the albumen gland, a female sex gland of the snail. In the present study calfluxin was purified and the amino acid composition was determined. The ratio of the amino acids appeared to be very close to that of one of the predicted peptides present on the egg-laying hormone precursor of Lymnaea. This peptide was synthesized and shows a clear biological activity in the bioassay. Furthermore, it shows a chromatographic behavior similar to that of the natural peptide. Based on these evidences it is concluded that the sequence of calfluxin is: Arg-Val-Asp-Ser-Ala-Asp-Glu-Ser-Asn-Asp-Asp-Gly-Phe-Asp. Calfluxin shows a remarkable homology with the sequence of one of the predicted peptides on the egg-laying hormone precursor of the marine opisthobranch Aplysia californica.

Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide.

Insulin and related peptides are key hormonal integrators of growth and metabolism in vertebrates. There is little biochemical evidence for insulin-related peptides in invertebrates, apart from insects for which definitive structural information on these peptides (prothoracicotropic hormone, PTTH) has recently been obtained. We report here the first complete complementary DNA-derived primary structure of a preproinsulin-related protein from identified neurons in an invertebrate, the mollusc Lymnaea stagnalis. We have demonstrated by in situ hybridization that transcription of the gene for this molluscan insulin-related peptide (MIP) occurs in the cerebral light-green cells, giant neuroendocrine cells involved in the control of growth, as well as in a pair of neuroendocrine cells called the canopy cells. The insulin-related peptide precursor has the same overall structure as its vertebrate counterparts. The discovery of insulin-related peptides in invertebrates substantiates the evidence for a widespread and early evolutionary origin of the insulin superfamily.

The brain of Lymnaea contains a family of FMRFamide-like peptides.

Authentic FMRFamide and two FMRFamide-related heptapeptides were purified from the central nervous system of the fresh water snail Lymnaea stagnalis. The sequences of the heptapeptides were determined as: Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide) by modified Edman degradation and enzymatic digestion. Relatively high quantities of the deamidated and therefore non-immunoreactive analogs of these two peptides (SDPFLRF and GDPFLRF) were also found. SDPFLRFamide and GDPFLRFamide were synthesized and were found to be chromatographically and biologically indistinguishable from the natural peptides, confirming the sequences. The log dose-response curves for the chronotropic action of either synthetic peptide on the heart of Lymnaea was very similar to that of FMRFamide. These data indicate that Lymnaea contains a family of FMRFamide-like peptides.

Purification and amino acid sequence of the ovulation neurohormone of Lymnaea stagnalis.

The neurosecretory caudodorsal cells of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation hormone [caudodorsal cell hormone (CDCH)] that is stored and released at the periphery of the intercerebral commissure. In the present study, CDCH has been purified and sequenced by micromethods. CDCH has been isolated, starting with a hydrochloric acid extract of commissures, by chromatofocusing, by high-performance, gel-permeation chromatography, and by reversed-phase, high-performance liquid chromatography. This procedure resulted in a 1690-fold purification and a 66% recovery. The data of the sequence analysis of CDCH are in agreement with the amino acid composition and reveal the following sequence of 36 amino acids: H-Leu-Ser-Ile-Thr-Asn-Asp-Leu-Arg-Ala-Ile-Ala-Asp-Ser-Tyr-Leu-Tyr-Asp-Gln-His -Trp-Leu-Arg-Glu-Arg-Gln-Glu-Glu-Asn-Leu-Arg-Arg-Arg-Phe-Leu-Glu-Leu-OH. Enzyme data indicate that the COOH end of the hormone is amidated. CDCH has a calculated isoelectric point of 9.0 and a calculated M(r) of 4529. CDCH shares a 44% homology with the sequence of the egg-laying hormone of the marine opisthobranch mollusc Aplysia californica.

Synthesis of multiple peptides from a larger precursor in the neuroendocrine caudo-dorsal cells of Lymnaea stagnalis.

The biosynthesis, transport and release of multiple peptides by the egg-laying controlling neuroendocrine caudo-dorsal cells (CDCs) of Lymnaea stagnalis were studied. High-performance gel permeation chromatography was used to resolve newly synthesized peptides after pulse-chase experiments with radioactive amino acids. The ultimate precursor is a approximately 35 kd (K) peptide which is produced in the CDC somata. It gives rise to intermediate products (approximately 20 K, approximately 10 K and approximately 7 K) and a number of end products which include a approximately 4.5 K peptide (the ovulation hormone) and other peptides (approximately 6 K, approximately 3.5 K and approximately 2 K). The end products are transported in neurosecretory granules to the CDC axon terminals in the cerebral commissure where they are released into the medium during electrical discharges of the CDC system.

Molecular properties of various snail peptides from brain and gut.

Attention is focused on the similarities in primary structure of the egg-laying neurohormone of the pulmonate Lymnaea stagnalis and of the opisthobranch Aplysia californica which both consist of 36 amino acid residues. FMRFamide-like peptides have now been isolated and sequenced from six molluscan species. Besides FMRFamide, two closely related peptides were isolated from the central nervous system of L. stagnalis and sequenced. This indicates that a family of FMRFamide-like peptides exist not only in the molluscs, but also within one species. A molluscan growth hormone, isolated from the brain of L. stagnalis, has been characterized. This small peptide hormone stimulates in vitro a receptor-adenylate cyclase system of mantle edge cells and in vivo the Ca2+-incorporation in the shell edge. The biochemical characterization of three vertebrate-like peptides of L. stagnalis, resembling oxytocin, Arg-vasopressin, and insulin, confirms the immunological findings that gastropods contain peptides which are structurally closely related to mammalian peptides.

Partial purification and characterization of the ovulation hormone of the freshwater pulmonate snail Lymnaea stagnalis.

The neurosecretory caudodorsal cells in the cerebral ganglia of the basommatophoran snail Lymnaea stagnalis produce an ovulation hormone (CDCH), which is released from the periphery of the intercerebral commissure. In the present study CDCH has been partially purified, starting with a hydrochloride acid extract of commissures, by gel permeation chromatography on Bio-Gel P-6 followed by cation exchange on SP-Sephadex C-25. This two-step procedure resulted in a ca. 22-fold purification of CDCH. The low purification factor is caused by poor resolution of both purification steps and low recovery on the Bio-Gel P-6 column. With stability tests, proteolytic enzymes, SDS/urea polyacrylamide electrophoresis and isoelectric focussing, purified CDCH was characterized as a stable peptide with a molecular weight of ca. 4700 d and a pI of ca. 9.3. The properties of CDCH are similar to those of the ovulation hormones of opisthobranch snails. Studies on species specificity indicate, however, that it is unlikely that CDCH will cause ovulation when injected in opisthobranchs, or vice versa.

Rapid separation of cytosol and particle fraction of human platelets by digitonin-induced cell damage.

Current cell disruption and fractionation techniques are time consuming and unsuitable for metabolic studies. We have developed a rapid method for platelets in which separation of cytosol and particle fraction is obtained within 50 s. Isolated platelet suspensions were incubated with low concentrations of digitonin followed by separation of soluble and particle fraction by centrifugation through a phthalate layer. Cell disruption was 90.1+/-4.2% (mean+/-SD, n=18; lactate dehydrogenase leakage). Contamination of granules: acid hydrolase vesicles 16.2+/-3.6% (n=18, beta-N-acetylglucosaminidase), dense granules 7--9% (n=3, 14C-serotonin), mitochondrial matrix 0.6+/-0.1% (n=18, glutamate dehydrogenase). Low concentrations of digitonin did not affect sialic acid content, nucleoside diphosphate kinase and phosphodiesterase activity in isolated membranes. The method showed that most enzymes of glycolysis and hexose monophosphate shunt were localized in the cytosol except for hexokinase (96% particle bound), phosphoglucose isomerase (10% bound) and glutathion reductase (26% bound). About half the total ATP+ADP and most glycolytic intermediates were found partly particle bound, especially fructose 1,6-diphosphate (40% bound). The data suggest that in platelets glycolysis occurs in different cell compartments.