Ingestion of food pellets containing Escherichia coli overexpressing the heat-shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection.

Feeding aquatic animals with bacterial encapsulated heat-shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK-DnaJ-GrpE, the prokaryotic equivalents of Hsp70-Hsp40-Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g(-1) pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.

Feeding Artemia franciscana (Kellogg) larvae with bacterial heat shock protein, protects from Vibrio campbellii infection.

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes.

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

Microtubule organization by cross-linking and bundling proteins.

To understand microtubule function the factors regulating their spatial organization and their interaction with cellular organelles, including other microtubules, must be elucidated. Many proteins are implicated in these organizational events and the known consequences of their actions within the cell are increasing. For example, the function of microtubule bundles at the surfaces of polarized cells has recently received attention, as has the action in cortical rotation of a transient arrangement of microtubules found beneath the vegetal surface of fertilized frog eggs. The in vivo association of microtubules during early Xenopus oogenesis has added interest as microtubules bundled in cell-free extracts are protected against the action of a severing protein found in this animal. A 52 kDa F-actin bundling protein purified from Physarum polycephalum organizes microtubules and causes the cobundling of microtubules and microfilaments. These observations, in concert with others that are presented, emphasize the diversity within the family of microtubule cross-linking proteins. The challenge is to determine which proteins are relevant from a physiological perspective, to ascertain their molecular mechanisms of action and to describe how they affect cytoplasmic organization and cell function. To realize this objective, the proteins which cross-link and bundle microtubules must be investigated by techniques which reveal different but related aspects of their properties. Cloning and sequencing of genes for cross-linking proteins, their subcellular localization especially as microtubule-related changes in cell morphology are occurring and the application of genetic studies are necessary. Study of the neural MAP provides the best example of just how powerful current experimental approaches are and at the same time shows their limits. The neural MAP have long been noted for their enhancement of tubulin assembly and microtubule stability. Their spatial distribution has been studied during the morphogenesis of neural cells. Sequencing of cloned genes has revealed the functional domains of neural MAP including carboxy-terminal microtubule-binding sites. Similarities to microtubule binding proteins from other cell types stimulate interest in the neural MAP and further suggest their importance in microtubule organization. For example, MAP4 enjoys a wide cellular distribution and has microtubule-binding sequences very similar to those in the neural MAP. Moreover, the nontubulin proteins of marginal bands are immunologically related to neural MAP, indicating shared structural/functional domains. Even with these findings the mechanism by which neural MAP cross-link microtubules remains uncertain. Indeed, some researchers express doubt that microtubule cross-linking is actually a function of neural MAP in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin.

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.

Cloning and sequencing of an alpha-tubulin cDNA from Artemia franciscana: evidence for translational regulation of alpha-tubulin synthesis.

The brine shrimp, Artemia franciscana, exhibits a limited number of tubulin isotypes which change little during early postgastrula growth. In order to better understand the synthesis of alpha-tubulins during Artemia development, a cDNA termed alphaAT1 was cloned and sequenced. Alignment analyses revealed that the polypeptide encoded by alphaAT1 is similar to alpha-tubulins from other species. Hybridization of alphaAT1 to restriction-digested DNA on Southern blots produced a simple banding pattern, indicating that Artemia have a small number of alpha-tubulin genes. Probing of Northern blots demonstrated an abundant supply of alpha-tubulin mRNA in dormant cysts, emerging nauplii and instar I larvae. However, it was not until instar I larvae were produced that the amount of polysomal alpha-tubulin mRNA increased, suggesting that synthesis of the tubulin corresponding to alphaAT1 is translationally controlled. This work provides one of the few examples where tubulin synthesis is thought to be translationally regulated. Moreover, when considered in the light of previous analyses, the findings imply that cell differentiation in postgastrula Artemia and the diversification of microtubule function certain to accompany this process occur with little or no change in alpha-tubulin composition.

Interphase cells of the centric diatom, Thalassiosira fluviatilis, lack detyrosinated, nontyrosinatable and acetylated tubulin.

Within eukaryotic cells tubulin generally exists in protein families composed of closely related isoforms generated either by differential gene transcription or by posttranscriptional and posttranslational mechanisms. In this study, immunological approaches were used to examine the contribution of posttranslational modifications to tubulin heterogeneity in a centric diatom, Thalassiosira fluviatilis, and to show the spatial distribution of microtubules in these cells during their interphase. After blotting to nitrocellulose, tubulin in cell-free homogenates of T. fluviatilis was recognized by several general tubulin antibodies including one to the tyrosinated isoform, but not by antibodies to detyrosinated, nontyrosinatable nor acetylated tubulins. Immunofluorescent staining of methanol-fixed cells revealed a net-like reticulum of microtubules originating at or near the cell nucleus. For all antibodies, except one (TU-01), results obtained by immunofluorescent experiments corrobated the analysis of blotted tubulins. Furthermore, microtubules exhibited differential staining patterns corresponding to the intensity of antibody reactivity on blots. Antibody to detyrosinated tubulin, as well as TU-01, yielded a spotty pattern of fluorescence on chloroplasts. Microtubules in T. fluviatilis support normal cell function in the absence of detectable amounts of three common posttranslationally modified tubulins, perhaps due to the rigid silica frustule which maintains shape and to the absence of flagella in interphase cells.

Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification.

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.

Temperature effects on solutions of vinblastine-induced polymers assembled from brine shrimp (Artemia) tubulin.

Purified Artemia tubulin in the presence of neural microtubule-associated proteins and vinblastine, or with vinblastine alone, forms extensive coils. Reduction in temperature of a coil-containing solution to 4 degrees C causes an increase in turbidity, which returns to previous levels once the solution is warmed. Examination of negatively stained samples indicates that the turbidity fluctuations are not accompanied by a pronounced change in coil structure nor by increased polymer formation. Bovine neural tubulin responds in the same way as Artemia tubulin to vinblastine and temperature. An interesting novel response to vinblastine, shared by tubulins from phylogenetically distinct organisms, is illustrated by our results.

Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding.

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana.

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.

Small heat shock proteins: molecular structure and chaperone function.

Small heat shock proteins (sHSPs) associate with nuclei, cytoskeleton and membranes, and as molecular chaperones they bind partially denatured proteins, thereby preventing irreversible protein aggregation during stress. sHSP monomers consist of a conserved alpha-crystallin domain of approximately 90 amino acid residues, bordered by variable amino- and carboxy-terminal extensions. The sHSPs undergo dynamic assembly into mono- and poly-disperse oligomers where the rate of disassembly affects chaperoning. The alpha-crystallin domain contains several beta-strands organized into two beta-sheets responsible for dimer formation, the basic building block of most sHSPS. The amino-terminal extension modulates oligomerization, subunit dynamics and substrate binding, whereas the flexible carboxy-terminal extension promotes solubility, chaperoning and oligomerization, the latter by inter-subunit linkage. Crystallization studies have revealed sHSP structure and function. Additionally, site-directed mutagenesis, biophysical investigations, functional studies and the discovery of relationships between mutated sHSPs and diseases have illuminated the role of sHSP within cells.

Bundling of bovine and brine shrimp (Artemia) microtubules in vitro.

Cell-free extracts from embryos of the brine shrimp (Artemia) induced bundling of bovine microtubules assembled in the presence of glycerol and Mg++. Sedimentation of microtubules through sucrose cushions and subsequent electrophoresis revealed that bundling occurred independently of accessory proteins tightly bound to the microtubules. Bovine microtubules containing microtubule-associated proteins (MAPS) or assembled with taxol did not bundle. The unusual polymerization properties of homogeneous Artemia tubulin, bundling in the absence of added factors and the small number of microtubules assembled in crude embryo extracts upon addition of taxol precluded a complete comparative study of Artemia and bovine microtubule bundling. Interesting properties of the in vitro assembly of Artemia microtubules were, however, elaborated and putative Artemia MAPs were observed as a consequence of the work with brine shrimp embryos.

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.

Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas.

Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.

Tubulin post-translational modifications--enzymes and their mechanisms of action.

This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosination/tyrosination, acetylation/deacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable alpha-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and alpha-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulin/microtubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications.

Developmental and comparative aspects of brine shrimp tubulin.

Tubulin from embryos of the brine shrimp Artemia has been purified to apparent homogeneity by chromatography on phosphocellulose P11 and DEAE-cellulose, (NH4)2SO4 fractionation and assembly-disassembly of microtubules. Peptide mapping indicated that Artemia and bovine brain tubulin were very similar in spite of differences in the electrophoretic behaviour of tubulin from these two organisms. Isoelectric focusing and two-dimensional gel electrophoresis were used to resolve and identify several Artemia isotubulins . The isotubulin composition and the quantity of tubulin did not change during pre-emergence development of Artemia embryos. Formation of microtubules with tubulin purified from embryos at different stages of development did not require glycerol or microtubule-associated proteins and formation of structurally normal microtubules was actually hindered by glycerol and Mg2+. The characteristics of Artemia tubulin, in concert with the unusual life history of Artemia, suggest that this organism will be very useful for the study of tubulin gene expression and tubulin utilization during embryo development.

Toxicity of organic mercury compounds to the developing brine shrimp, Artemia.

Mercury can be coupled to a wide variety of organic compounds but there is limited information concerning the influence of such substitutions on the toxicity of mercury within the marine environment. We therefore determined the effects of six organomercuries on the emergence and hatching of the brine shrimp, Artemia. The relative toxicities of the organic mercuries were unaffected by the ability of the compounds to ionize, whereas the sizes of the compounds appeared to be important. Thus, brine shrimp were equally sensitive to five of the organic mercuries while diphenylmercury, the largest of the organic mercuries tested, was the least toxic. In the presence of 0.1 microM diphenylmercury the final amount of hatching was similar to that in the absence of metal but in this situation there was an easily measured reduction in the rate of development. By determining the rates of emergence and hatching it is apparent that Artemia are adversely affected by organic mercuries at concentrations less than 0.1 microM, the lowest level examined in this study. The work extends our earlier findings with cadmium and zinc, supporting the proposal that Artemia is an excellent alternative to more complex, slow-growing animals for the study of biochemical/physiological aspects of marine pollution.

A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro.

A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5'-adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences.(ABSTRACT TRUNCATED AT 250 WORDS)