Hippocampal levels of gamma-enolase, C-1-tetrahydrofolate synthase and serotransferrin fluctuate over the estrous cycle in the rat.

Hippocampal functions vary across the estrous cycle but metabolic changes at the protein level have not been systematically studied so far. It was therefore the aim of the study to screen expression of metabolic proteins mainly represented by metabolic enzymes in the hippocampus over the estrous cycle and in males. Female and male OFA Sprague-Dawley rats were used and female estrous phases were determined by vaginal smears, according to which females were separated into groups of proestrous, estrous, early and late metestrous and diestrous. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis followed by identification with mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Comparative analysis of protein levels was carried out by quantifying protein spot volumes by means of specific software. Levels of one expression form of gamma-enolase were different between diestrous and early metestrous; C-1-tetrahydrofolate synthase levels were elevated in proestrous as compared with estrous and serotransferrin levels were increased in diestrous as compared with proestrous, estrous, metestrous and in males. The outcome of estrous cycle- and gender-dependent protein fluctuations is relevant for the interpretation of previous and future work as well as for the design of further studies at the protein level in the hippocampus.

Molecular cloning and expression of a bovine endothelial inward rectifier potassium channel.

A 5.1 kb cDNA encoding an inward rectifier K+ channel (BIK) was isolated from a bovine aortic endothelial cell library. The cDNA codes for a 427-amino-acid protein with two putative transmembrane regions. Sequence analysis reveals that BIK is a member of the Kir2.1 family of inward rectifier K+ channels. Expression in Xenopus oocytes showed that BIK is a K+-specific strong inward rectifier channel that is sensitive to extracellular Ba2+, Cs+, and a variety of anti-arrhythmic agents. Northern analysis revealed that endothelial cells express a 5.5 kb BIK mRNA that is sensitive to shear stress.

Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M1 receptor sensitivity in Xenopus oocytes.

Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, resulting in an increased formation of IP3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca2+ released from intracellular stores in response to IP3 then opens Ca(2+)-gated Cl-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT1c receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT1c receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was approximately 50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca(2+)-free superfusate indicated that desensitization of the 5-HT1c receptor response does not result from a sustained elevation of intracellular Ca2+ level or require the entry of extracellular Ca2+. Photolysis of caged IP3 demonstrated that an increase in IP3 and a subsequent rise in Ca2+ do not produce desensitization of either the IP3 or 5-HT1c peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M1 acetylcholine receptor, the M1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M1 current response and desensitization of the 5-HT1c current response may be the same.

Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA.

The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes.

Modulation of a cloned mouse brain potassium channel.

The mouse brain K+ channel (MBK), previously cloned by others, has been independently cloned and shown to express in Xenopus oocytes. This K+ current (IK) inactivated over a time course of seconds and was sensitive to the K+ channel-blocking reagent tetraethylammonium. When the K+ channel was coexpressed with a cloned mouse brain serotonin receptor (5HT1c) in oocytes, activation of the 5HT1c receptor by a brief application of serotonin resulted in a suppression of the IK amplitude over the next 20 min. IK could also be suppressed by activation of G proteins. Suppression was also caused by intracellular Ca2+ injections and was blocked by intracellular injection of EGTA. Calmodulin antagonists block the IK suppression, but a known protein kinase inhibitor did not block suppression. The 5HT1c suppression was reversible; recovery from suppression was blocked by the protein kinase inhibitor H-7. These data suggest that the IK suppression occurs through a novel mechanism independent of A- or C-type protein kinases; suppression is best explained as being due to the action of a Ca2+/calmodulin-activated phosphatase; recovery from suppression is due to the action of a protein kinase.

At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes.

Fast transient K+ channels (A channels) of the type operating in the subthreshold region for Na+ action potential generation were expressed in Xenopus oocytes injected with rat brain poly(A) RNA. Sucrose gradient fractionation of the RNA separates mRNAs encoding A-currents (6-7 kb) from mRNAs encoding other voltage-dependent K+ channels. A-currents expressed with fractionated mRNA differ in kinetics and pharmacology from A-currents expressed with total mRNA. The original properties of the A-currents can be reconstituted when small mRNAs (2-4 kb) are added to the large mRNA fraction. Thus the properties of the A-currents expressed with total poly(A) RNA depend on the presence of more than one mRNA species. mRNA(s) present in the large RNA fraction must encode channel subunits since they express an A-current by themselves. The small mRNA(s) may encode a second subunit(s) or a factor, such as an enzymatic activity that modulates the properties of the channels, which could play a role in generating A-channel functional diversity.

Membrane adenosine triphosphatase in synchronous cultures of Rhodobacter sphaeroides.

Studies of intracytoplasmic membrane biogenesis utilizing synchronized cultures of Rhodobacter sphaeroides have revealed that most intracytoplasmic membrane proteins accumulate continuously throughout the cell cycle while new phospholipid appears discontinuously within the intracytoplasmic membrane. The resulting changes in the structure of the membrane lipids was proposed to influence the activities of enzymes associated with the intracytoplasmic membranes (Wraight, C.A., Leuking, D.R., Fraley, R.T. and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). We have extended the study of intracytoplasmic membrane biogenesis in R. sphaeroides to include the membrane adenosine triphosphatase. The membrane bound Mg2+-dependent, oligomycin-sensitive adenosine triphosphatase activity was measured throughout the cell cycle for steady-state synchronized cells of R. sphaeroides and found to accumulate discontinuously. Following treatment with an uncoupling reagent (2,4-dinitrophenol) the intracytoplasmic membrane associated adenosine triphosphatase activity was stimulated uniformly in membranes isolated at different stages of the cell cycle. The adenosine triphosphatase was also measured by quantitative immunoblots utilizing specific antibody to compare the enzyme activity and enzyme protein mass. Immunologic measurement of the adenosine triphosphatase in isolated membranes indicated a constant ratio of enzyme to chromatophore protein exists during the cell cycle in contrast to the discontinuous accumulation of adenosine triphosphatase activity. These results are discussed in light of the cell-cycle specific synthesis of the intracytoplasmic membrane.

Cloning and expression of the Rhodobacter sphaeroides reaction center H gene.

The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragements specific for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane.

Phospholipid transfer activity in synchronous populations of Rhodobacter sphaeroides.

Studies of intracytoplasmic membrane biogenesis employing steady-state synchronously dividing populations of Rhodobacter sphaeroides reveal that the translocation of pre-existing phospholipid into the growing membrane is concurrent with cell division (Cain, B.D., Deal, C.D., Fraley, R.T. and Kaplan, S. (1981) J. Bacteriol. 145, 1154-1166), yet the mechanism of phospholipid movement is unknown. However, the discovery of phospholipid transfer protein activity in R. sphaeroides (Cohen, L.K., Lueking, D.R. and Kaplan, S. (1979) J. Biol. Chem. 254, 721-728) provides one possible mechanism for phospholipid movement. Therefore the level of phospholipid transfer activity in cell lysates of synchronized cultures was measured and was shown to increase stepwise coinciding precisely with the increase in cell number of the culture. Although the amount of transfer activity per cell remained constant throughout the cell cycle, the specific activity of the phospholipid transfer activity showed a cyclical oscillation with its highest value coincident with the completion of cell division. Purified intracytoplasmic membrane can be used as phospholipid acceptor in the developed phospholipid transfer assay by employing either cytoplasmic membrane or liposomes as the phospholipid donor. Intracytoplasmic membrane isolated from the cells prior to division (high protein to phospholipid ratio) served as a better phospholipid acceptor in the phospholipid transfer system when compared with membranes derived from the cells following cell division (low protein to phospholipid ratio).

In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides.

The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo- with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.

Topology and neighbor analysis of the photosynthetic reaction center from Rhodopseudomonas sphaeroides.

The subunit arrangement of the reaction center complex (RC) of Rhodopseudomonas sphaeroides was studied by chemical modification with four different cross-linking reagents using purified RC in lauryldimethylamine oxide, RC incorporated into liposomes, and intact chromatophore membranes, from which RCs are isolated. The RC of R. sphaeroides is composed of three polypeptide subunits, H, M, and L, apparent molecular mass as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 28,000, 24,000, and 21,000, respectively. The intra-complex products produced, were found to contain the polypeptides H-M-L, H-M, H-L, and M-L linked together. In addition, the cross-linking of cytochrome c to solubilized and membrane-bound RCs was observed with all four reagents. The products were found to be only a cytochrome c linked to either the M or L polypeptide. These results indicate that a portion of the L and M subunits of the RC must be exposed in situ on the periplasmic surface of the membrane near a binding site for cytochrome c on the RC, and all three subunits must be in close proximity to one another.

Characterization of three intermediates in the biosynthesis of teichuronic acid of Micrococcus luteus.

Teichuronic acid, the Micrococcus luteus cell wall polysaccharide which consists of D-glucose and N-acetyl-D-mannosaminuronic acid, is synthesized in vitro from uridine diphosphate N-acetyl-D-glucosamine, uridine diphosphate N-acetyl-D-mannosaminuronic acid, and uridine diphosphate D-glucose in a series of reactions catalyzed by a particulate enzyme preparation. Several lipid-linked intermediates are formed, of which the first three are called components A, B, and C. The formation of these intermediates is inhibited by tunicamycin. The lipid moiety of the intermediates is approximately 95% undecaprenol and 5% dodecaprenol as determined by mass spectrometry. The oligosaccharide moieties of components B and C are the disaccharide, N-acetyl-D-mannosaminuronyl-(1,3)-N-acetyl-D-glucosamine, and the trisaccharide, N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminuronyl++ +-(1, 3)-N-acetyl-D-glucosamine, respectively, as determined by the complete degradation of the former and partial degradation of the latter by the alkaline beta-elimination reaction. The saccharide and lipid moieties of the intermediates are linked through pyrophosphate. Thus, component A is P1-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, component B is P1-N-acetyl-D-mannosaminuronyl-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, and component C is P1-N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminurony l-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate.