Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.     

Cuticular proteins from the horseshoe crab,Limulus polyphemus

Proteins were purified from the carapace cuticle of a juvenile horseshoe crab, Limulus polyphemus, and several of them were characterized by amino acid sequence determination. The proteins are small (7-16 kDa) and their isoelectric points range from 6.5 to 9.2. They have high contents of tyrosine, ranging from 13.5 to 35.4%. Some of the proteins show sequence similarity to cuticular proteins from other arthropod groups, with the most pronounced similarity to proteins from the cuticle of the spider Araneus diadematus. Two proteins show sequence similarity to a hexamerin storage protein from Blaberus discoidalis.     

Spermiogenesis in the horseshoe crab,Limulus polyphemus

Groups of spermatids of Limulus polyphemus undergo differentiation in thin-walled cysts within the seminiferous tubules. The nucleus compacts to a spherical shape, but retains a much less condensed nuclear appendage, whose unique pores are each surrounded by a microtubule. The appendage, unmodified mitochondria, glycogen, and coated vesicles, all present in the mature spermatozoon, suggest an unusual degree of metabolic self-sufficiency of the cell. The acrosome is associated with a 50 μ-long acrosomal filament that penetrates the nucleus during spermiogenesis and coils up in the cytoplasm, enveloped by two outer nuclear membranes. The filament, which eventually comes to lie in the circumnuclear cisterna, retains a covering of one membrane during its discharge at the time of the acrosome reaction. The posterior region of the head forms a thin-walled collar with peculiar internal supports around the base of the flagellum. Serverance of intercellular bridges between spermatids, cytoplasm elimination, and rupture of the cyst precede liberation of the immature spermatozoa into the lumen of the seminiferous tubules. Notwithstanding its peculiarities, the Limulus spermatozoon, with its simple shape closely resembling that of annelids and molluscs, represents the most primitive arthropod spermatozoon congruent with the evolutionary stability of the xiphosurans.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.     

The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus.

Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development. Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus. Semaphorins are divided into eight classes according to species of origin and structural similarities. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are present in vertebrates and viruses encode class V semaphorin. Microarray analysis of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus polyphemus. Based on these data, we aligned 31 different sequences and designed degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW). RT-PCR products were generated using 6 forward primers and 4 reverse primers. The expected size PCR products (750 bp) was obtained and then ligated with pCR II TOPO vector and transferred into E. coli Top 10. Five partial semaphorin cDNAs were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were partially cloned. Subsequent Northern blot analyses using these Limulus specific-probes revealed hybridization with total RNAs purified from six different tissues.     

Bactericidal activity of granules isolated from amoebocytes of the horseshoe crab, Limulus polyphemus

Granules were isolated from amoebocytes of Limulus polyphemus and used in bactericidal studies. Whole granules, but not granular lysates, exhibited marked killing activity against Escherichia coli and Staphylococcus aureus. These findings support the idea that amoebocytes are an important component in the defense system of this animal.     

Presence of microorganisms in hemolymph of the horseshoe crab Limulus polyphemus.

Hemolymph samples obtained from Limulus polyphemus at the time of collection and after a 1-week holding period exhibited a significant increase in bacterial levels. No differences were observed in the ability of amoebocyte lysate, prepared from these same samples, to gel in the presence of lipopolysaccharide.     

An atlas of the brain of the horseshoe crab Limulus polyphemus

An atlas of the brain of the horseshoe crab Limulus polyphemus is developed. All of the neuronal groups are identified and named, and regions of neuropil are segregated and named where possible. The nomenclature incorporates functionally neutral earlier names and assigns geographical names to newly distinguished structures. The atlas provides a basis for correlating the results of neuroanatomical, neurophysiological, and neurochemical studies, which yield information about individual neurons or groups of neurons in this species.     

A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.     

Estimation of total hemolymph volume in the horseshoe crab Limulus polyphemus

Biomedical companies extract blood from the horseshoe crab, Limulus polyphemus, for the production of Limulus Amebocyte Lysate, used worldwide for detecting endotoxins in injectable solutions and medical devices. Despite the extensive use of horseshoe crabs by the biomedical industry, total hemolymph volume for this species is not known. The hemolymph volume of 60 adult horseshoe crabs was estimated using an inulin dilution technique. Blood volume of the horseshoe crab represented as a percentage of wet body weight was 25?±?2.2% for males and 25?±?5.1% (mean?±?SD) for females. Relationships between hemolymph volume and weight (p?=?0.0026, r ²?=?0.8762), hemolymph volume and prosomal width (p?<?0.0001), and hemolymph volume and inter-ocular width (p?<?0.0001) were observed. No significant differences were observed between males and females. The relationship of animal size and hemolymph volume can be used to predict how much blood can be drawn from horseshoe crabs used by the biomedical industry, and can be of further use in future bleeding mortality studies.     

Vitamin B12-binding proteins of the horseshoe crab Limulus polyphemus

Vitamin B12-binding proteins were detected in the body fluids and/or tissues of horseshoe crabs (Limulus polyphemus), clams and sponges. Among the biological specimens tested the Limulus plasma was especially rich in free B12-binding proteins. Gel filtration experiments revealed that Limulus plasma contains two classes of B12-binding proteins. One class of proteins, molecular weight in excess of 100,000, bind B12 preferentially with affinity constant of 5 X 10(11)M-1. The second type of proteins, molecular weights around 50,000, bind B12 with specificity approaching that of mammalian intrinsic factors. The binding constant of these proteins for B12 is around 10(11)M-1.     

The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus

We determined the complete 14,985-nt sequence of the mitochondrial DNA of the horseshoe crab Limulus polyphemus (Arthropoda: Xiphosura). This mtDNA encodes the 13 protein, 2 rRNA, and 22 tRNA genes typical for metazoans. The arrangement of these genes and about half of the sequence was reported previously; however, the sequence contained a large number of errors, which are corrected here. The two strands of Limulus mtDNA have significantly different nucleotide compositions. The strand encoding most mitochondrial proteins has 1. 25 times as many A's as T's and 2.33 times as many C's as G's. This nucleotide bias correlates with the biases in amino acid content and synonymous codon usage in proteins encoded by different strands and with the number of non-Watson-Crick base pairs in the stem regions of encoded tRNAs. The sizes of most mitochondrial protein genes in Limulus are either identical to or slightly smaller than those of their Drosophila counterparts. The usage of the initiation and termination codons in these genes seems to follow patterns that are conserved among most arthropod and some other metazoan mitochondrial genomes. The noncoding region of Limulus mtDNA contains a potential stem-loop structure, and we found a similar structure in the noncoding region of the published mtDNA of the prostriate tick Ixodes hexagonus. A simulation study was designed to evaluate the significance of these secondary structures; it revealed that they are statistically significant. No significant, comparable structure can be identified for the metastriate ticks Rhipicephalus sanguineus and Boophilus microplus. The latter two animals also share a mitochondrial gene rearrangement and an unusual structure of mt-tRNA(C) that is exactly the same association of changes as previously reported for a group of lizards. This suggests that the changes observed are not independent and that the stem-loop structure found in the noncoding regions of Limulus and Ixodes mtDNA may play the same role as that between trnN and trnC in vertebrates, i.e., the role of lagging strand origin of replication.     

Presence of microorganisms in hemolymph of the horseshoe crab Limulus polyphemus

Hemolymph samples obtained from Limulus polyphemus at the time of collection and after a 1-week holding period exhibited a significant increase in bacterial levels. No differences were observed in the ability of amoebocyte lysate, prepared from these same samples, to gel in the presence of lipopolysaccharide.     

Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus)

Often referred to as a living fossil, the American horseshoe crab, Limulus polyphemus, is one of the most-studied invertebrate animals in the world. It has served as a model in Nobel Prize-winning eye research, and researchers use a component of its blood to detect bacterial contamination in medical devices and drugs. The authors review the conditions necessary for housing these animals in the laboratory.     

Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.     

Cortical reaction in inseminated eggs of the horseshoe crab,Limulus polyphemus L

The egg cortical reaction in Limulus polyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smooth-appearing surface approximately 60–90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg—smooth surface, (II) inseminated egg—appearance and growth of pits, (III) inseminated egg—coalescence of pits, and (IV) inseminated egg—appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique.     

Histamine metabolism in the visual system of the horseshoe crab Limulus polyphemus

There is now strong evidence that arthropod photoreceptors use histamine as a neurotransmitter. The synthesis, storage and release of histamine from arthropod photoreceptors have been demonstrated, and the postsynaptic effects of histamine and the endogenous neurotransmitter are similar. However, a full understanding of these photoreceptor synapses also requires knowledge of histamine inactivation and metabolism. Relatively little is known about histamine metabolism in the nervous system of arthropods, and mechanisms appear to differ with the species. This study focuses on histamine metabolism in visual tissues of the horseshoe crab Limulus polyphemus, a chelicerate. We present two major findings: (1) histamine is metabolized to imidazole acetic acid and to gamma-glutamyl histamine. (2) relatively low levels of histamine metabolites accumulate in Limulus visual tissues.