The effects of the main body cations on the peptidase(s) activity against pyroGlu6[125I-Tyr8]SP6-11 in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain

1. Peptidase(s) activity of nuclear and synaptosomal fraction from cortex and hippocampus of rat brain against pyroGlu6[125I-Tyr8]SP6-11 was evaluated in different concentration of Ca2+, Mg2+, K+ and Na+ in about "isotonic" conditions. 2. The effects of studied ions on the peptidase activities forming N-terminal and C-terminal fragments are different especially in synaptosomes of both areas. 3. The differences of ionic requirements for N- and C-forming activities are particularly relevant for Ca2+ at the cortex and K+ at the hippocampus. 4. Ca2+ activate forming of N-terminal fragments in the nuclear fraction whereas inhibit it in synaptosomes from both areas. 5. The ionic requirements for C-terminal fragments' formation in synaptosomes of both areas are contradictory.     

Molecular biology of cell activation

This article summarizes common features of activation of different types of cells along different physiological lines such as proliferation, differentiation, and execution of function of terminally differentiated cells. The common basis of many of these phenomena includes (i) first messengers (growth factors, cytokines, neurotransmitters, etc.) acting on membrane receptors, (ii) second messengers (cAMP, IP3, DAG, Ca2+) spreading an activating signal inside the cell, and (iii) elevated expression of some genes (c-fos, c-myc, ornithine decarboxylase). The role of the genetic correlate in cell activation is emphasized, and it is concluded that the aforementioned genes (their protein products) should be called third messengers, whose function is mediation of long-term phenotypic changes.     

The influence of second messengers and related substances on the sensitivity of early embryos to cytostatic neurochemicals

"Prenervous" neurotransmitters interact with second messengers in the regulation of early embryogenesis of sea urchins and the clawed frog Xenopus laevis. Propranolol and atropine inhibit cleavage divisions of X. laevis only after their intracellular administration. The level of the membrane potential (including a zero level) of the embryos studied is not essential for the cleavage divisions. Possible mechanisms of action of the "prenervous" neurotransmitters at the intracellular level and their coupling with second messengers are discussed.     

The mature size of rat 4-aminobutyrate aminotransferase is different in liver and brain.

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of beta-AlaAT I (4-aminobutyrate aminotransferase, beta-alanine-oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34-amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of beta-AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western-blot analysis, mass spectroscopy and N-terminal sequencing. The mature form of beta-AlaAT I from the brain had an ISQAAAK- peptide on the N-terminus of the liver mature beta-AlaAT I. Brain beta-AlaAT I was cleaved to liver beta-AlaAT I when incubated with fresh mitochondrial extract from rat liver. These results imply that mature rat liver beta-AlaAT I is proteolytically cleaved in two steps. The first cleavage of the motif XRX( downward arrow)XS is performed by a mitochondrial processing peptidase, yielding an intermediate-sized protein which is the mature brain beta-AlaAT I. The second cleavage, which generates the mature liver beta-AlaAT I, is also carried out by a mitochondrial endopeptidase. The second peptidase is active in liver but lacking in brain.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.     

A second look at the second messenger hypothesis

Several hundred hormones, neurotransmitters, growth factors and other "first messengers" bind to specific cell membrane receptors and induce a myriad of effects: short term, transport, metabolic, mitotic and regulation of thousands of specific genes. Yet, less than a dozen "second messengers" have been clearly established to date. Even allowing for the discovery of a large number of additional second messengers, there remains a paradox in terms of information-transfer within the cell: how can so many specific signals produce so many effects through so few relatively nonspecific intermediates? We consider several possible solutions to this paradox, including the hypothesis that signal specificity is encoded in part in the primary structure of the receptor.     

Non-kinase second-messenger signaling: new pathways with new promise

Intercellular signaling by growth factors, hormones and neurotransmitters produces second messenger molecules such as cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG). Protein Kinase A and Protein Kinase C are the principal effector proteins of these prototypical second messengers in certain cell types. Recently, novel receptors for cAMP and DAG have been identified. These proteins, designated EPAC (Exchange Protein directly Activated by cAMP) or cAMP-GEF (cAMP regulated Guanine nucleotide Exchange Factor) and CalDAG-GEF (Calcium and Diacylglycerol regulated Guanine nucleotide Exchange Factor) or RasGRP (Ras Guanine nucleotide Releasing Protein) are able to mediate some of the physiologic effects of the second messengers in a protein-kinase-independent fashion. These proteins are exchange factors for Ras family GTPases that operate in pathways that run parallel to the classic kinase-dependent pathways. The rapidly emerging recognition of the functions of these "non-kinase" effectors in diverse processes such as insulin secretion, thymocyte development, asthma and malignant transformation creates new opportunities for discovery and identifies potential new therapeutic targets.     

Genetic complementation in yeast reveals functional similarities between the catalytic subunits of mammalian signal peptidase complex

Type I signal peptidases (SPs) comprise a family of structurally related enzymes that cleave signal peptides from precursor proteins following their transport out of the cytoplasmic space in eukaryotic and prokaryotic cells. One such enzyme, the mitochondrial inner membrane peptidase, has two catalytic subunits, which recognize distinct cleavage site motifs in their signal peptide substrates. The only other known type I SP with two catalytic subunits is the signal peptidase complex (SPC) in the mammalian endoplasmic reticulum. Here, we tested the hypothesis that, as with inner membrane peptidase catalytic subunits, SPC catalytic subunits exhibit nonoverlapping substrate specificity. We constructed two yeast strains without endogenous SP, one expressing canine SPC18 and the other expressing a truncation of canine SPC21 (SPC21 Delta N), which lacks 24 N-terminal residues that prevent expression of SPC21 in yeast. By monitoring a variety of soluble and membrane-bound substrates, we find that, in contrast to the tested hypothesis, SPC catalytic subunits exhibit overlapping substrate specificity. SPC18 and SPC21 Delta N do, however, cleave some substrates with different efficiencies, although no pattern for this behavior could be discerned. In light of the functional similarities between SPC proteins, we developed a membrane protein fragmentation assay to monitor the position of the catalytic sites relative to the surface of the endoplasmic reticulum membrane. Using this assay, our results suggest that the active sites of SPC18 and SPC21 Delta N are located 4-11 A above the membrane surface. These data, thus, support a model that SPC18 and SPC21 are functionally and structurally similar to each other.     

Intracellular substrates for extracellular signaling. Characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin)

We previously identified (Sobel, A., and Tashjian, A. H., Jr. (1983) J. Biol. Chem. 258, 10312-10324) a group of cytoplasmic proteins whose phosphorylation could be related to the regulation by extracellular effectors of cells as different as pituitary and muscle cells. Among these phosphoproteins, proteins "7" and "8" (Mr approximately 19,000, pI approximately 5.8-6.0), that we now designate P1 and P2, are very abundant in rat brain. Partial purification of these proteins was therefore achieved after 100 degrees C precipitation of a rat brain-soluble fraction and further fractionation of the supernatant by ion exchange chromatography. Several related non-phosphorylated (N1, N2) and phosphorylated (P3) proteins were also identified in the heat-resistant supernatant. Antisera raised against P2 extracted from nitrocellulose blots of semipreparative two-dimensional gels recognized all the proteins N1, N2, P1, P2, and P3, confirming that they belong to the same protein family, and suggesting that they are likely various forms of a single protein core. The same protein could be detected biochemically and immunologically at various concentrations in all the tissues or cell types from diverse mammalian and nonmammalian species tested. Together with our previous data relating its phosphorylation to the regulation of the proliferation, differentiation, and/or the functions of the cells considered, this observation leads us to suggest that it might be an ubiquitous regulatory phosphoprotein playing the role of an intracellular "relay" for extracellular signals, after their binding to specific membrane receptors and the generation of second messengers. We propose to name this protein stathmin, from the greek "stathmos" (relay).     

The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization

Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization.     

The preliminary evaluation of degradation of substance P(SP) fragment's analogue less than Glu SP6-11 in the subcellular fractions from different areas of rat brain

Peptidase(s) activity of different subcellular fractions isolated from cortex, hippocampus, midbrain, thalamus with hypothalamus, cerebellum and medulla oblongata exerted against less than Glu SP6-11 (3H-Phen8) was evaluated in "low-ionic" and similar (in composition) to both extracellular and intracellular conditions. The incubation of less than Glu SP6-11 with different fractions leaves the hexapeptide undegraded in the studied conditions in most cases. Peptidases activity results in the formation of the first of all C-terminal and exceptionally "internal" labelled products. Labelled N-terminal products were not seen. The most effective degradation in vitro of less than Glu SP6-11 takes place, in the majority of cases, in "low ionic" conditions when compared to those similar to extra or intracellular ones. The biggest total (per 1 g of wet mass) and specific activities against less than Glu SP6-11 can be shown in the hippocampus areas.     

The Croonian lecture, 1988. Inositol lipids and calcium signalling

The response of cells to many external stimuli requires a decoding process at the membrane to transduce information into intracellular messengers. A major decoding mechanism employed by a variety of hormones, neurotransmitters and growth factors depends on the hydrolysis of a unique inositol lipid to generate two key second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here I examine the second messenger function of Ins(1,4,5)P3 in controlling the mobilization of calcium. We know most about how this messenger releases calcium from internal reservoirs but less is known concerning the entry of external calcium. One interesting possibility is that Ins(1,4,5)P3 might function in conjunction with its metabolic product Ins(1,3,4,5)P4 to control calcium entry through a mechanism employing a region of the endoplasmic reticulum as a halfway house during the transfer of calcium from outside the cell into the cytoplasm. The endoplasmic reticulum interposed between the plasma membrane and the cytosol may function as a capacitor to insure against the cell being flooded with external calcium. When stimulated, cells often display remarkably uniform oscillations in intracellular calcium. At least two oscillatory patterns have been recognized suggesting the existence of separate mechanisms both of which may depend upon Ins(1,4,5)P3. In one mechanism, oscillations may be driven by periodic pulses of Ins(1,4,5)P3 produced by receptors under negative feedback control of protein kinase C. The other oscillatory mechanism may depend upon Ins(1,4,5)P3 unmasking a process of calcium-induced calcium release from the endoplasmic reticulum. The function of these calcium oscillations is still unknown. This Ins(1,4,5)P3/calcium signalling system is put to many uses during the life history of a cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin

An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7-Phe8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCI pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 75 HR 10/30) using a new preparative system, AKTA-purifier, controlled by UNICORN software version 2.20.     

Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

Phospholipids and electrolyte transport.

Our understanding of the role of phospholipids in ion transport processes is only beginning to be appreciated. Although the role of polyphosphoinositide and its derived second messenger molecules IP3, diacylglycerol, and arachidonic acid are well studied, we are still not certain as to how changes in the lipid bilayer structure influence the status of ion channels. This review focused on those studies which show a strong correlation with ion conductance changes and the status of the membrane phospholipids. In addition, a number of observations point to a major role of lipid second messengers that activate enzymes involved in protein phosphorylations, i.e., protein kinase C, as major regulators of a variety of ion channels and transporters. Such lipid second messengers provide a cellular mechanism whereby hormones, neurotransmitters, and pharmacologic agents functionally control the ionic environment and intracellular pH of target cells. Some of these pathways still remain to be elucidated; however, an appreciation for the participation of membrane phospholipids in these actions has been presented.     

Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.