Temporal appearance and distribution of the Ca2+ + Mg2+ ATPase of the sarcoplasmic reticulum in developing chick myocardium as determined by immunofluorescence labeling

The temporal appearance and distribution of the Ca2+ + Mg2+ ATPase of the sarcoplasmic reticulum were determined in the developing chick heart (stage 9 to stage 16) by indirect immunofluorescence labeling. The results obtained showed that the Ca2+ + Mg2+ ATPase was first observed in the bulbus ventricular region of the single tubular heart at stage 9 to 10 of development, when these myocardial cells first contract. As the atrial and later the sinus venosus tissues became incorporated into the single tubular heart the Ca2+ + Mg2+ ATPase was also observed in these regions, however, the highest density of Ca2+ + Mg2+ ATPase labeling was generally observed in the region of the heart most recently incorporated. These results suggest that the sarcoplasmic reticulum is present and perhaps functional in the regulation of the cytoplasmic Ca2+ concentration and thereby the contraction-relaxation cycle in myocardial cells when the first contraction occurs, as well as throughout all subsequent stages of development. Furthermore comparison between the relative density and intensity of the Ca2+ + Mg2+ ATPase labeling and the intrinsic rate of contraction of the myocardial cells in the various regions of the heart (A. Barry, 1942, J. Exp. Zool. 91, 119-130) supports the possibility that a positive correlation exists between these two characteristics of the myocardial cells.     

Amplification and deletion of the amyE+-tmrB+ gene region in a Bacillus subtilis recombinant-phage genome by the tmrA7 mutation.

A 22.4-kilobase DNA fragment containing the tmrA7-amyR2-amyE+-tmrB+-aroI+ region of the Bacillus subtilis N7 chromosomal DNA was cloned into a recombinant B. subtilis bacteriophage, p11-AA248. The amyE+-tmrB+ gene region, approximately 12.6 kilobases, in the phage genome was amplified in a tunicamycin-resistant (Tmr) Amy+ AroI+ transductant of B. subtilis by p11-AA248. On the other hand, the amyE+-tmrB+ region in the genomes of 80 to 90% of the phage particles was deleted when the phages were induced from the Tmr Amy+ AroI+ transductants by treatment with 1.0 micrograms of mitomycin C per ml. From analyses of the physical maps and DNA nucleotide sequences in the junction region of the deleted phage genome and the parental DNA fragments, it is suggested that the deletion occurred within a direct repeat sequence composed of 18 base pairs. The endpoints of the amplified gene region seemed to be closely related to both terminal regions of the deleted DNA.     

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.     

Cytogenetic localization of a putative regulatory gene affecting an NADP+-dependent enzyme in Drosophila melanogaster

The cytogenetic localization of a putative regulatory locus affecting an NADP+-dependent enzyme is described. This locus, Mex, is in region 8D10-12 to 9A1-2 on the X chromosome of Drosophila melanogaster. Hyperploidy for this region is associated with significant increases in NADP+-dependent malic enzyme specific activity and specific immunologically cross-reacting material. This is the first report of a specific locus, unlinked to the locus coding for the major polypeptide of the enzyme, which affects an NADP+-dependent enzyme in Drosophila melanogaster.     

Microdomain arrangement of the SERCA-type Ca2+ pump (Ca2+-ATPase) in subplasmalemmal calcium stores of paramecium cells.

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of approximately 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( approximately 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

The action of trypsin on the gastric (H+ + K+)-ATPase.

The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis.     

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum

In the heart, excitation-contraction (E-C) coupling is mediated by Ca²⁺ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca²⁺ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca²⁺ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202–231). Second, in vitro binding assays were conducted to examine the Ca²⁺ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca²⁺ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca²⁺ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca²⁺ into SR at intermediate Ca²⁺ concentrations.     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

Mg2+-, Ca2+-dependent unwinding of DNA by poly-L-glutamic acid

In order to examine a possibility that the high acidic amino acid region in the nonhistone protein HMG(1+2) is concerned with the Mg2+-, or Ca2+-dependent unwinding of DNA by the HMG(1+2) (1,2), poly-L-glutamic acid was employed as an acidic model peptide for thermal melting temperature analysis. The poly-L-glutamic acid bound to DNA either in the presence or absence of Mg2+. The poly-L-glutamic acid unwound DNA double-helix to a similar extent to HMG(1+2) in the presence of Mg2+ or Ca2+, but not in the absence of them. These results may suggest that the high acidic amino acid region in HMG(1+2) participates in Mg2+-, Ca2+-dependent unwinding of DNA double-helix.     

CTLA-4+49A>G基因多态性与结直肠癌的相关性研究

目的:探讨细胞毒性T淋巴细胞相关抗原-4(CTLA-4)+49 AG位点多态性与结直肠癌发生的相关性,为早期预测结直肠癌的发生提供临床参考依据。方法:选取结直肠癌病例231例未实验组和正常健康体检者325例为对照组,取其空腹外周静脉血提取DNA后,采用聚合酶链式反应(PCR)技术对CTLA-4基因第1外显子区+49位点DNA进行扩增,产物用限制性片段长度多态性(RFLP)方法检测CTLA-4第1外显子区+49AG位点的多态性,比较两组杂合子AG和纯合子AA、GG基因型发生的频率、A等位基因与G等位基因的分布,分析CTLA-4+49 AG位点多态性与结直肠癌发生的相关性。结果:两组间CTLA-4基因外显子1区+49AG位点杂合子AG和纯合子AA、GG基因型发生的频率以及A等位基因与G等位基因的分布比较差异均无统计学意义(P0.05)。结论:CTLA-4基因外显子1区+49 AG基因多态性与我省汉族人群结直肠癌的发病无显著相关性。     

Activity coefficients of the peptide and the electrolyte in ternary systems water+glycylglycine+NaCl, +NaBr, +KCl and +KBr at 298.2 K

The activity coefficients of glycylglycine in four aqueous electrolyte solutions (+NaCl, +NaBr, +KCl and +KBr) were obtained at 298.2 K. The mean ionic activity coefficient of the electrolyte in aqueous solutions containing the peptide was determined from measurements of the potential differences of a cation and an anion ion-selective-electrode, each vs. a double junction reference electrode. The results show that the nature of the anion has a major effect on the activity coefficients of glycylglycine. Comparison of activity coefficient data for glycylglycine with literature data for glycine, both in aqueous NaCl solutions, indicates that the effect of the electrolyte is larger for the peptide than for the amino acid. For the peptide, in all cases, the effect of the electrolyte is more important at low molalities of the electrolyte. The Wilson equation was used to correlate the activity coefficient data obtained. The correlation results were satisfactory for the region of concentrated electrolyte.     

Expression and regulation of H+K+ATPase in lysosomes of epithelial cells of the adult rat epididymis

Endocytosis is an important event in the epididymis as it contributes to a luminal environment conducive for sperm maturation. Principal and clear cells contain numerous lysosomes which degrade many substances internalized by endocytosis from the epididymal lumen. The interior of the lysosomes depends on low pH to activate the release of their enzymes and to activate their acid hydrolases. In the present study, H+K+ATPase was localized by light microscopy in the adult rat epididymis of intact and of orchidectomized animals supplemented with testosterone or not. In normal animals, numerous lysosomes of nonciliated cells of the efferent ducts were intensely reactive for anti-H+K+ATPase antibody. In the initial segment, only a few lysosomes of principal cells were reactive. In the intermediate zone of the epididymis, numerous lysosomes of principal cells were intensely reactive, while the number of intensely reactive lysosomes decreased progressively from the proximal caput to the distal caput with none being seen in the proximal corpus region. In the distal corpus and cauda regions, only a few lysosomes of some principal cells were reactive. In contrast, clear cells of all regions showed intense reactivity. Orchidectomy resulted in the abolishion of H+K+ATPase in lysosomes of principal cells of all regions except the initial segment. However, while clear cells of the caput and corpus regions also became unreactive, those of the cauda region remained as reactive as in controls. Orchidectomized animals supplemented with testosterone maintained a staining pattern similar to controls for both cell types. These observations demonstrate the presence in principal and clear cells of H+K+ ATPase which may have an important role in acidifying the interior of their lysosomes. However, there is a region-specific expression of H+K+ATPase in lysosomes of principal cells, unlike that for clear cells. In addition, H+K+ATPase expression in lysosomes of principal cells depends on testosterone in all regions except the initial segment. However, in the case of clear cells, only those of the caput and the corpus regions are dependent on testosterone, while those of the cauda region appear to be regulated by some other factor.