Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.     

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.     

Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis.

In previous studies we and others have described several mitochondrial proteins which are synthesized in response to acute hormone stimulation in several steroidogenic tissues. In both MA-10 mouse Leydig tumor cells and primary cultures of rat adrenal cortex cells, these proteins consist of a family of 37 kilodalton (kDa) and 32 kDa precursor forms and fully processed forms which are 30 kDa in molecular weight. The nature of the appearance of these proteins and their subcellular localization to the mitochondria, the site of the rate limiting step in steroidogenesis, has led to the speculation that they may be involved in the acute regulation of steroidogenesis. In the present study we have taken advantage of another steroidogenic cell, the R2C rat Leydig tumor cell, to perform studies which further indicate that these mitochondrial proteins are involved in the regulation of steroidogenesis. Unlike the MA-10 cell which requires hormone stimulation for steroid production, the R2C cell is a constitutive progesterone producer whose steroid production cannot be further increased with hormone stimulation. We have shown that the R2C cell line is less sensitive to the inhibition of steroid production by the metal chelator orthophenanthroline (OP) than is the MA-10 cell. We have demonstrated that progesterone production and the 30 kDa mitochondrial proteins remain present in the R2C cells at a concentration of OP which completely inhibits progesterone production and totally eliminates the 30 kDa proteins in MA-10 cells. As further evidence for the role of these proteins in steroidogenic regulation, we have isolated several revertants of the R2C parent (P) cell line which have lost the ability to synthesize progesterone constitutively, but which can be stimulated to synthesize this steroid by trophic hormone and cAMP analog. In these revertants, designated (R), the normally constitutively present 30 kDa proteins are greatly decreased compared to controls, but reappear in large amounts following hormone stimulation. Taken together, these data provide further evidence that the 30 kDa mitochondrial proteins are involved in the acute regulation of steroidogenesis in Leydig cells.     

Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle

Developmental regulation, from the fetal period to 11 months of age, and the influence of denervation on the appearance and disappearance of the molecular forms of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) in rat skeletal muscle were examined. The enzyme forms were extracted from anterior tibialis in 0.01 M sodium phosphate buffer, pH 7.0, containing 1 N NaCl, 0.01 M EGTA, 1% Triton X-100, and a cocktail of antiproteases, and analyzed by velocity sedimentation on 5-20% linear sucrose gradients. Three principal forms, denoted by sedimentation coefficients of 4, 10.8, and 16 S, were observed in muscle from all age groups. The amounts of each of the molecular forms of AchE and BuchE in skeletal muscle exhibited distinct and reciprocal patterns of appearance and disappearance during pre- and postnatal development. In tissue derived from animals less than 2 weeks of age, BuchE represented the predominant component of activity in the 4 S form, was present equally with AchE in the 10.8 S form, and was subordinate to AchE in the 16 S form. Between 1 and 2 weeks of age a progressive increase in AchE activities coincident with a reduction in BuchE activities resulted in inversion in the amounts of the two enzymes present in adult muscle. Denervation of muscle caused a dramatic reduction in the presence of AchE molecular forms with no discernable influence on the presence of BuchE molecular forms. These results indicate that biosynthesis of BuchE is strictly regulated in a reciprocal manner with that of AchE, and that BuchE metabolism is independent of the state of muscle innervation. Increased synthesis of AchE and either reduced synthesis or increased degradation of BuchE can account for the reciprocal regulation of these enzymes. These characteristics of mammalian muscle contrast sharply with characteristics deduced for avian tissue (Silman et al. (1979) Nature (London) 280, 160-162). The innervation-independent metabolism of BuchE and the diverse modes of its regulation in different tissue from different species signify that BuchE function may be unrelated to cholinergic neurotransmission.     

Proteolytic modification of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-512F dextransucrase

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209–Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.Martha Argüello-Morales and Mónica Sánchez-González equally contributed to this work.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Characterization of mouse serum tenascin-X

The interstitial extracellular matrix tenascin-X (iTNX), which has a molecular mass of roughly 450 kDa, is expressed at high levels in muscular tissues and skin. In this study, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in the mouse. Western blot analysis with specific antibodies against fibronectin type III-like (FNIII) repeats of TNX and N-terminal sequence analysis of 200-kDa sTNX revealed that the N-terminus of sTNX is located in the juncture between the 16th FNIII (M16) and 17th FNIII (M17) repeats of iTNX. The 200-kDa sTNX contains 15 FNIII repeats and a fibrinogen domain identical to the Cterminal portion of the iTNX. TNX-deficient mice lacked not only iTNX but also sTNX. Furthermore, 200-kDa sTNX was generated by cleavage of the spleen iTNX by spleen homogenate, and its generation was inhibited by protease inhibitors. These results suggest that sTNX is generated by proteolytic cleavage of iTNX.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.     

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.     

Biochemical properties of alkaline phosphatase from endometrial cancer cells

The occurrence of alkaline phosphatase (AP) activity was examined in several human endometrial adenocarcinomas. Catalytic activities were detectable only in 10 out of 15 tumors, with no apparent correlation between elevated AP and histological type. The apparent molecular weight of the enzyme after partial purification was about 140,000 daltons. Kinetic activity, thermodynamic properties and the pH dependence of the activity were in the ranges reported for other subforms. Several other physicochemical properties were also investigated and compared with those displayed by enzymes obtained from normal human tissues. The inhibition studies show that the enzyme shares several properties with the placental form, particularly in resistance to zinc chloride and EDTA action. On the other hand, in sensitivity to uncompetitive inhibitors and to urea and ascorbic acid, it is closer to other non-Regan heat-sensitive forms. The results support the view that a polymorphism in the expression of AP in neoplastic tissues can occur. A wider spectrum of physicochemical properties is clearly needed to define better the characteristics of oncodevelopmental enzymes.     

Developmentally regulated proteolytic processing of a yolk glycoprotein complex in embryos of the sea urchin,Strongylocentrotus purpuratus

We have isolated a yolk glycoprotein complex from eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of these complexes and peptide mapping of their individual glycoprotein components indicate that developmental stage-specific changes in molecular composition of the complex are due to proteolytic processing events. Our data revealed that a 180 kDa glycoprotein of the egg complex is separated by a single proteolytic cleavage into intermediate glycoproteins of 115 and 76 kDa early in development. By the hatched blastula stage, each of these intermediate glycoproteins has been further processed to lower molecular weight forms: the 115 kDa protein is proteolytically clipped to a 84 kDa form, perhaps through 110 and 105 kDa intermediaries, while the 76 kDa molecule is directly processed to a 65 kDa form.     

beta1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T) expression in normal rat tissues and different cell lines: evidence for complex mechanisms of regulation

The distribution of the Golgi enzyme beta1, 6-N- acetylglucosaminyltransferase (core 2 GlcNAc-T for short) has been investigated in several tissue and cell systems by combining the potentials of a polyclonal antibody and a novel, sensitive fluorescent enzyme assay. In normal rat tissues, levels of the protein were found to vary and as a general trend did not correlate with enzyme activities. Additionally, we observed tissue-specific core 2 GlcNAc-T forms of various size: 75 kDa (liver), 70 kDa (spleen), 60 kDA (heart), and 50 kDa (heart and lung). These forms might arise from differential protein modifications; alternatively, the smaller form may be a product of proteolytic cleavage, given the presence of a catalytically inactive 50 kDa species in rat serum. Chinese hamster ovary (CHO), MDAY-D2, PSA- 5E, and PYS-2 cell lines consistently displayed a 70 kDa enzyme. When induced to retrodifferentiate in the presence of butyrate + cholera toxin, CHO cells exhibited a 21-fold increase in enzyme activity, while protein levels remained constant. A similar trend was observed in the embryonal endoderm cell lines PSA-5E and PYS-2, where an approximately 100-fold difference in core 2 GlcNAc-T activity was found notwithstanding unchanged amounts of the protein and identical mRNA levels, as evidenced by RT-PCR. In contrast, levels of core 2 GlcNAc-T activity in MDAY-D2 cells correlated well with protein expression. Taken together, these observations demonstrate that core 2 GlcNAc-T expression may be subjected to multiple mechanisms of regulation and suggest that in at least some instances (i.e., PSA-5E and PYS-2 cells) expression may be regulated exclusively via posttranslational mechanism(s) of control.      

Comparative studies on the primary structure of acetylcholinesterases from bovine caudate nucleus and bovine erythrocytes

1. Comparison of partial amino acid sequences of G2-acetylcholinesterase (AChE) from bovine erythrocytes and G4-AChE from bovine caudate nucleus revealed no differences in primary structure between the two enzymes. The first 33 residues of the N-terminal sequences were identical. 2. In addition, the amino acid sequences of four peptides generated by tryptic and cyanogen bromide cleavage were identical for bovine erythrocyte and brain AChE, suggesting one identical major coding exon for the adult bovine AChE forms. Comparison of these sequences with that of fetal bovine serum AChE (Doctor et al., 1988), showed differences in residues 16, 181, 212, and 216. 3. Deglycosylation studies of the two adult enzyme forms revealed that the core protein of erythrocyte AChE has an approximately 4 kDa lower molecular mass than brain AChE. This most probably reflects differences in the C-terminal sequences of the two enzymes.     

A cathepsin D-cleaved 16 kDa form of prolactin mediates postpartum cardiomyopathy

Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.     

Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family

A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family. It shares several biochemical and molecular properties with the clan ME family M16 metallopeptidases (pitrilysins), as well as with database hypothetical proteins that are potential M16 family enzymes. Thus, like insulysin and nardilysin, but contrary to bacterial pitrilysin, ppBH4 is inactivated by sulfhydryl alkylating agents. On the other hand, like bacterial pitrilysin, ppBH4 is sensitive to reducing agents. The enzymatic activity of ppBH4 is limited to substrates smaller than proteins. In contrast to insulin, dynorphin and insulin B-chain are very good substrates for ppBH4 and several cleavage sites are common with those observed with well-characterized pitrilysins. As deduced from amino acid sequence, as well as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis, ppBH4 is an active monomer of 46.5 kDa. This feature distinguishes ppBH4 from all other enzymes of the pitrilysin family so far described whose molecular masses range from 100 to 140 kDa.     

Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues.

We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species.     

Molecular signals of plasticity at the tetrapartite synapse

The emergence of astroglia as an important participant of the synaptic machinery has led to the 'tripartite synapse' hypothesis. Recent findings suggest that synaptic signaling also involves the surrounding extracellular matrix (ECM). The ECM can incorporate and store molecular traces of both neuronal and glial activities. It can also modulate function of local receptors or ion channels and send diffuse molecular signals using products of its use-dependent proteolytic cleavage. Recent experimental findings implicate the ECM in mechanisms of synaptic plasticity and glial remodeling, thus lending support to the 'tetrapartite synapse' concept. This inclusive view might help to understand better the mechanisms underlying signal integration and novel forms of long-term homeostatic regulation in the brain.