Human and mouse albumin bind their respective neonatal Fc receptors differently.

Albumin has a serum half-life of three weeks in humans and is utilized to extend the serum persistence of drugs that are genetically fused or conjugated directly to albumin or albumin-binding molecules. Responsible for the long half-life is FcRn that protects albumin from intracellular degradation. An in-depth understanding of how FcRn binds albumin across species is of importance for design and evaluation of albumin-based therapeutics. Albumin consists of three homologous domains where domain I and domain III of human albumin are crucial for binding to human FcRn. Here, we show that swapping of two loops in domain I or the whole domain with the corresponding sequence in mouse albumin results in reduced binding to human FcRn. In contrast, humanizing domain I of mouse albumin improves binding. We reveal that domain I of mouse albumin plays a minor role in the interaction with the mouse and human receptors, as domain III on its own binds with similar affinity as full-length mouse albumin. Further, we show that P573 in domain III of mouse albumin is required for strong receptor binding. Our study highlights distinct differences in structural requirements for the interactions between mouse and human albumin with their respective receptor, which should be taken into consideration in design of albumin-based drugs and evaluation in mouse models.

Genetic coding algorithm for sense and antisense peptide interactions.

Sense and antisense peptides, i.e. peptides specified by complementary DNA and RNA sequences, interact with increased probability. Biro, Blalock, Mekler, Root-Bernstein and Siemion investigated the recognition rules of peptide-peptide interaction based on the complementary coding of DNA and RNA sequences in 3'→5' and 5'→3' directions. After more than three decades of theoretical and experimental investigations, the efficiency of this approach to predict peptide-peptide binding has been experimentally verified for more than 50 ligand-receptor systems, and represents a promising field of research. The natural genetic coding algorithm for sense and antisense peptide interactions combines following elements: of amino acid physico-chemical properties, stereochemical interaction, and bidirectional transcription. The interplay of these factors influences the specificity of sense-antisense peptide interactions, and affects the selection and evolution of peptide ligand-receptor systems. Complementary mRNA codon-tRNA anticodon complexes, and recently discovered Carter-Wolfenden tRNA acceptor-stem code, provide the basis for the rational modeling of peptide interactions based on their hydrophobic and lipophilic amino acid physico-chemical properties. It is shown that the interactions of complementary amino acid pairs according to the hydrophobic and lipophilic properties strongly depend on the central (second) purine base of the mRNA codon and its pyrimidine complement of the tRNA anticodon. This enables the development of new algorithms for the analysis of structure, function and evolution of protein and nucleotide sequences that take into account the residue's tendency to leave water and enter a nonpolar condensed phase considering its mass, size and accessible surface area. The practical applications of the sense-antisense peptide modeling are illustrated using different interaction assay types based on: microscale thermophoresis (MST), tryptophan fluorescence spectroscopy (TFS), nuclear magnetic resonance spectroscopy (NMR), and magnetic particles enzyme immunoassay (MPEIA). Various binding events and circumstances were considered, e.g., in situations with-short antisense peptide ligand (MST), L- and D-enantiomer acceptors (TFS), in low affinity conditions (NMR), and with more than one antisense peptide targeting hormone (MPEIA).

Feedback inhibition of ammonium uptake by a phospho-dependent allosteric mechanism in Arabidopsis.

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time- and concentration-dependent manner. Neither Gln nor l-methionine sulfoximine-induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).

A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

Subthalamic stimulation increases striatal tyrosine hydroxylase phosphorylation.

Subthalamic stimulation enhances striatal tyrosine hydroxylase activity, which is regulated by phosphorylation at different serine residues. Western blotting was performed to investigate phosphorylation at the serine residues 19, 31 and 40 in striatal tissue of rats that had received subthalamic stimulation or sham stimulation for 2 h. In animals that were killed directly after stimulation, the tyrosine hydroxylase protein content was unchanged, whereas phosphorylation at the serine residue 19 was increased and phosphorylation at the serine residues 31 and 40 tended to be higher compared with controls. By contrast, tyrosine hydroxylase protein content and phosphorylation were similar in rats that were killed 24 h after stimulation. Our results suggest that subthalamic stimulation may increase tyrosine hydroxylase activity via increased phosphorylation.

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus.

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val(1)]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells.

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.

Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment.

Bcl-2 family member Bid is subject to autoinhibition; in the absence of stimuli, its N-terminal region sequesters the proapoptotic Bcl-2 homology 3 (BH3) domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C). Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly but not exclusively in helix 1, which is required for ubiquitination and degradation of tBid-N. Rescue of tBid-N from degradation blocked Bid's ability to induce mitochondrial outer membrane permeability but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord.

26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.

A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family.

BACKGROUND: Kazal-like serine protease inhibitors are defined by a conserved sequence motif. A typical Kazal domain contains six cysteine residues leading to three disulfide bonds with a 1-5/2-4/3-6 pattern. Most Kazal domains described so far belong to this class. However, a novel class of Kazal domains with two disulfide bridges resulting from the absence of the third and sixth cysteines have been found in biologically important molecules, such as human LEKTI, a 15-domain inhibitor associated with the severe congenital disease Netherton syndrome. These domains are referred to as atypical Kazal domains. Previously, EPI1, a Kazal-like protease inhibitor from the oomycete plant pathogen Phytophthora infestans, was shown to be a tight-binding inhibitor of subtilisin A. EPI1 also inhibits and interacts with the pathogenesis-related P69B subtilase of the host plant tomato, suggesting a role in virulence. EPI1 is composed of two Kazal domains, the four-cysteine atypical domain EPI1a and the typical domain EPI1b. RESULTS: In this study, we predicted the inhibition constants of EPI1a and EPI1b to subtilisin A using the additivity-based sequence to reactivity algorithm (Laskowski algorithm). The atypical domain EPI1a, but not the typical domain EPI1b, was predicted to have strong inhibitory activity against subtilisin A. Inhibition assays and coimmunoprecipitation experiments showed that recombinant domain EPI1a exhibited stable inhibitory activity against subilisin A and was solely responsible for inhibition and interaction with tomato P69B subtilase. CONCLUSION: The finding that the two disulfide bridge atypical Kazal domain EPI1a is a stable inhibitor indicates that the missing two cysteines and their corresponding disulfide bond are not essential for inhibitor reactivity and stability. This report also suggests that the Laskowski algorithm originally developed and validated with typical Kazal domains might operate accurately for atypical Kazal domains.

The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel.

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Identification of neurite extension inducing peptides by means of soluble combinatorial peptide libraries.

To identify hexapeptides capable of inducing neurite outgrowth, we used three groups of soluble combinatorial peptide libraries each consisting of 100 mixtures of hexapeptides (each mixture consisting of 10,000 individual peptides) with partially predetermined sequences (in two out of six amino acid positions). Using this approach a number of neuritogenic peptides were identified. Three selected peptides, QSGKKF, QSGPLA and QSGKQG, were found to induce neurite outgrowth from primary hippocampal neurons with potency comparable to that of growth factors. None of the peptides protected cerebellar granule neurons from cell death induced by withdrawal of potassium chloride. The approach described here suggests the feasibility to use combinatorial peptide libraries in order to identify compounds capable of modulating a specific functional response in the nervous system, without prior knowledge of a molecular target.

Molecular determinants of two neurotoxins that regulate sodium current inactivation in rat hippocampal neurons.

We studied functional and structural differences between the two neurotoxins, wasp toxin pompilidotoxin (PMTX) and sea anemone toxin (ATXII). Although PMTX and ATXII inhibited inactivation of sodium currents both toxins had distinct actions on the lobster axon and on the rat hippocampal cells. To determine structural basis of the difference we compared arrangement of polar and non-polar amino acids of the two toxins and found that similar sequence of PMTX exist in a discrete position of three-dimensional structure of ATXII. The sequence may be responsible for the binding site in the neuronal Na(+) channel molecule because PMTX is insensitive to cardiac Na(+) channel. Differential actions of ATXII from PMTX may come from other regions than the overlapped sequence. PMTX has diverse actions in the central neurons and is useful to classify Na(+) channel subtypes.

Modulation of delayed-type hypersensitivity responses in hairless guinea pigs by peptides derived from enkephalin.

Although opioid peptides such as methionine (met)-enkephalin have been previously shown to enhance or suppress immune responses, few studies in animal models have addressed the immunomodulatory activity of their metabolic derivatives. Hairless (IAF/HA-HO) guinea pigs immunized with Freund's complete adjuvant containing Mycobacterium tuberculosis and repeatedly skin tested with purified protein derivative of tuberculin (PPD) display high levels of stable delayed-type hypersensitivity (DTH) to PPD. Met-enkephalin (YGGFM) and two of its metabolites (YGG, YG) enhanced and accelerated PPD-elicited DTH inflammatory reactions when injected together with elicitor in these animals. At 24 h, 5 x 10(-3) pmol met-enkephalin significantly enhanced DTH responses by 30% over PPD alone, while 5 x 10(-5) pmol of YGG and 5 x 10(-9) pmol of YG significantly enhanced these responses by 62 and 32%, respectively. At much higher doses (5 x 10(3) pmol), met-enkephalin and its metabolites significantly suppressed DTH reactions by 25-32%. Tyrosine and glycine had no effect on PPD-elicited DTH. All DTH reactions (control, enhanced, suppressed) displayed typical perivascular mononuclear cell infiltrates. We conclude that the immunoactivity of met-enkephalin resides in its first two amino acids and suggest that cleavage of enkephalin molecules to YG occurs in serum and/or on the cell surface.

The netrin-G1 ligand NGL-1 promotes the outgrowth of thalamocortical axons.

Netrin-G1 is a lipid-anchored protein that is structurally related to the netrin family of axon guidance molecules. Netrin-G1 does not bind any of the known netrin receptors and its function is not known. Here we identify human netrin-G1 ligand (NGL-1), a transmembrane protein containing leucine-rich repeat (LRR) and immunoglobulin (Ig) domains that specifically interacts with netrin-G1 through its LRR region. Whereas netrin-G1 is expressed highly in mouse thalamic axons, NGL-1 is most abundant in the striatum and the cerebral cortex--the intermediate and final targets, respectively, of thalamocortical axons (TCAs). Surface-bound NGL-1 stimulates, but soluble NGL-1 disrupts, the growth of embryonic thalamic axons, and in vitro data indicate that NGL-1 activity may be mediated at least partially by netrin-G1. Our findings provide evidence that netrin-G1 functions as an important component of the NGL-1 receptor to promote TCA outgrowth and that membrane-bound netrins can participate in receiving axonal signaling pathways.

Improved brain delivery of benzylpenicillin with a peptide-vector-mediated strategy.

Previous studies from our laboratory have demonstrated that the coupling of doxorubicin with SynB1 vector dramatically increases its brain uptake. In the present study, we have evaluated the broad application of this approach using another molecule: benzylpenicillin (B-Pc). We, therefore, have coupled the beta-lactam antibiotic B-Pc with SynB1 and assessed its ability to cross the blood-brain barrier (BBB) using the in situ rat brain perfusion method. We first confirmed the very low brain uptake of free radiolabeled B-Pc. When B-Pc was coupled to SynB1, its uptake in brain was increased by a factor of 7, without compromising the BBB integrity. The vectorised B-Pc was distributed in all the gray areas assessed (frontal, parietal, and occipital cortex, thalamus, hippocampus, and striatum). Moreover, using a wash-out procedure and a capillary depletion method, we have shown that the radiolabeled B-Pc was associated mainly with brain parenchyma. In summary, this study demonstrates the successful application of the use of SynB1 vector for the transport of B-Pc across the BBB.

Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the brain of the European green frog Rana esculenta.

A novel neuropeptide of the RFamide peptide family was isolated in pure form from a frog (Rana esculenta) brain extract by using reversed-phase high performance liquid chromatography in combination with a radioimmunoassay for mammalian neuropeptide FF (NPFF). The primary structure of the peptide was established as Ser-Leu-Lys- Pro-Ala-Ala-Asn-Leu-Pro-Leu- Arg-Phe-NH(2). The sequence of this neuropeptide, designated Rana RFamide (R-RFa), exhibits substantial similarities with those of avian LPLRFamide, gonadotropin-inhibitory hormone, and human RFRP-1. The distribution of R-RFa was investigated in the frog central nervous system by using an antiserum directed against bovine NPFF. In the brain, immunoreactive cell bodies were primarily located in the hypothalamus, i.e., the anterior preoptic area, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. The most abundant population of R-RFa-containing neurons was found in the periependymal region of the suprachiasmatic nucleus. R-RFa- containing fibers were widely distributed throughout the brain from the olfactory bulb to the brainstem, and were particularly abundant in the external layer of the median eminence. In the spinal cord, scattered immunoreactive neurons were found in the gray matter. R-RFa-positive processes were found in all regions of the spinal cord, but they were more abundant in the dorsal horn. This study provides the first characterization of a member of the RFamide peptide family in amphibians. The occurrence of this novel neuropeptide in the hypothalamus and median eminence and in the dorsal region of the spinal cord suggests that, in frog, R-RFa may exert neuroendocrine activities and/or may be involved in the transmission of nociceptive stimuli.

Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis.

The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.

Carboxyl terminal peptides derived from prepro-orphanin FQ/nociceptin (ppOFQ/N) are produced in the hypothalamus and possess analgesic bioactivities.

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand for the ORL-1/KOR-3 receptor, produces a wide variety of behavioral responses. Its precursor protein, prepro-OFQ/N (ppOFQ/N) contains several series of amino acids bounded by pairs of basic amino acids, raising the possibility that additional functional neuropeptides could be generated by proteolytic posttranslational processing. Several of these processing products have been shown to have pharmacological activity, including the 17 amino acid peptide OFQ/N (mppOFQ/N(140-157)) which is a major product of this precursor in the hypothalamus. Here we have used a newly developed radioimmunoassay and RP-HPLC to detect mppOFQ/N(160-187) in mouse hypothalamic extracts. Murine ppOFQ/N(160-187) has potent analgesic activity supraspinally (3.4 nmol, i.c.v.) and spinally (4.3 nmol, i.t.). This analgesic activity was reversed by the opioid antagonist naloxone (5 mg/kg, s.c.) and kappa(1)-selective opioid antagonist nor-BNI (60 microg, i.c.v.), despite the inability of ppOFQ/N(160-187) to compete binding in mu, delta, kappa(1), kappa(3), or OFQ/N binding assays. These findings suggest that murine ppOFQ/N(160-187) may be a physiologically relevant neuropeptide with a novel mechanism of action.

Primary structure of myosin from the striated adductor muscle of the Atlantic scallop, Pecten maximus, and expression of the regulatory domain.

We have determined the complete cDNA and deduced amino acid sequences of the heavy chain, regulatory light chain and essential light chain which constitute the molecular structure of myosin from the striated adductor muscle of the scallop, Pecten maximus. The deduced amino acid sequences of P. maximus regulatory light chain, essential light chain and heavy chain comprise 156, 156 and 1940 amino acids, respectively. These myosin peptide sequences, obtained from the most common of the eastern Atlantic scallops, are compared with those from three other molluscan myosins: the striated adductor muscles of Argopecten irradians and Placopecten magellanicus, and myosin from the siphon retractor muscle of the squid, Loligo pealei. The Pecten heavy chain sequence resembles those of the other two scallop sequences to a much greater extent as compared with the squid sequence, amino acid identities being 97.5% (A. irradians), 95.6% (P. magellanicus) and 73.6% (L. pealei), respectively. Myosin heavy chain residues that are known to be important for regulation are conserved in Pecten maximus. Using these Pecten sequences, we have overexpressed the regulatory light chain, and a combination of essential light chain and myosin heavy chain fragment, separately, in E. coli BL21 (DE3) prior to recombination, thereby producing Pecten regulatory domains without recourse to proteolytic digestion. The expressed regulatory domain was shown to undergo a calcium-dependent increase (approximately 7%) in intrinsic tryptophan fluorescence with a mid-point at a pCa of 6.6.