Reconstitution of ATP-dependent calcium transport from streptococci

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.     

Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94-98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pI of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell-free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross-reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.     

夏季高温对水牛瘤胃代谢的影响

利用南京地区夏季炎热的自然条件,连续两年在高温季节(7—8月)进行实验。第一年(系列Ⅰ)的实验动物为四头装置瘤胃瘘管的空怀母水牛,研究高温初期(27.5～33.4℃)和持续高温期(28.0～35℃)对水牛瘤胃消化代谢的影响。第二年实验(系列Ⅱ)利用三头装置瘤胃瘘管的海仔母水牛重复高温(26～35.3℃)实验。 夏季高温期间,实验水牛的呼吸率、瘤胃温度和直肠温度升高,采食量减少,饮水量增加,瘤胃液流速减缓。高温初期出现瘤胃代谢升高[总挥发性脂肪酸(TVFA)和氨氮(NH_3-N)浓度及乙酸/丙酸(A/P)比率升高]。但在持续高温情况下,水牛的采食和瘤胃代谢均明显抑制。采取瘤胃内降温措施(投入冰袋)或冷水淋浴,均能迅速降低呼吸率、直肠和瘤胃温度,恢复采食和反刍,并缓解瘤胃代谢的抑制。提示动物机体参与调节瘤胃代谢的变化,并为改善水牛夏季的饲养管理提供生理学依据。     

北京东郊作物土壤系统中重金属的迁移、分布、积累

 本文研究了北京东郊污灌区重金属在作物—土壤中的迁移、分布、积累规律,证实本区蔬菜中汞含量比粮食作物约大3—15倍,比水果约大6—200倍。麦粒、糙米中的Cu、Hg、Cd、Pb、Ni的含量与土壤含量相关性不显著。架豆中重金属含量与土壤中重金属含量的相关性,只有Zn,Pb达显著水平。白菜土有机质含量与重金属含量相关性达显著水平,而白菜的重金属含量与土壤的重金属含量相关性却不显著。说明除了土壤中重金属的总量外,有效态含量的多少,是影响本区作物吸收积累重金属的主要因素。 本区施污泥的土壤和生长的作物Cd／Zn大部小于1%、盆栽试验证明：施用本区污泥污水对水稻生长发育的影响比施污泥灌清水的影响大些,因此,施用含重金属污泥时,最好不要超过5000斤／亩。大田和室内模拟试验证明：重金属从土壤中迁移到植物,由植物带走输出的量极少,其中以带走输出的Hg、Cd,As相对较多,带走输出的Pb、Cr相对的少些。     

苜蓿二磷酸核酮糖(RuBP)羧化酶体内活化作用的调节

苜蓿RuBP羧化酶的初活性和活化作用在不饱和光强下与光合速率一样随光强增加而增加。缺硫培养苜蓿叶片的光合速率和RuBP羧化酶的含量、初活性及总活性均比对照有不同程度的降低,其中酶的初活性与光合速率两者减少的趋势比较接近,说明RuBP羧化酶的初活性可能在光合CO_2固定作用中具有决定作用。然而,缺硫植株中酶的活化作用比对照明显增高。酶的活化作用与叶片中的叶绿素,6-PG,NADPH及ATP相对酶含量的比值成正比,与体内的酶量成反比。     

Hormonal regulation of dopa decarboxylase during a larval molt

Cuticular sclerotization in insects requires dopamine derivatives and thus the presence of dopa decarboxylase (DDC), the enzyme which converts dopa to dopamine. During the last half of the larval molt of the tobacco hornworm, Manduca sexta, beginning at 16 hr after head capsule slippage, the epidermal DDC activity increased fourfold. By contrast, allatectomized larvae which were destined to produce a melanized cuticle showed a sevenfold increase. This increase in DDC activity was prevented by infusion of 20-hydroxyecdysone (20HE) into the larva, indicating that the fall of the ecdysteroid titer is necessary for the increase. In vitro 20HE also prevented the increase in a dose-dependent manner when the epidermis was explanted at 16 hr after head capsule slippage but had less effect on epidermis explanted 3 hr later. Both 5 micrograms/ml alpha-amanitin and 100 micrograms/ml cycloheximide also prevented the increase. Application of juvenile hormone I showed that the critical period for determination of the level of the later increase in DDC activity was about 4 hr after head capsule slippage at the peak of the ecdysteroid titer. Apparently then the rise and fall of ecdysteroid regulate different aspects of DDC synthesis, the rise determining its later appearance and the fall timing this appearance.     

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodem GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.     

Purification and properties of creatinine iminohydrolase from Flavobacterium filamentosum

Creatinine iminohydrolase (EC 3.5.4.21), which catalyzes hydrolysis of creatinine to N-methylhydantoin and ammonia, was purified from Flavobacterium filamentosum. The average molecular weight of the purified enzyme was 272,480, and the subunit molecular weight was 44,300. Extensive specificity studies indicated that the enzyme utilized cytosine (Km, 0.62 mM; Vm, 20.1 units/mg) as well as creatinine (Km, 5.00 mM; Vm, 40.4 units/mg) as a substrate. Each was a competitive inhibitor toward hydrolysis of the other compound. Dialysis of creatinine iminohydrolase in the presence of 0.01 M Tris phosphate buffer, pH 7.5, containing 1,10-phenanthroline decreased activity by 98%. Reactivation was accomplished by incubating the apoenzyme in the presence of certain divalent metal chlorides, listed in decreasing order of effectiveness: iron(II), zinc, cobalt(II), cadmium, and nickel. The extent of reactivation depended on the substrate and on which metal ion was added to the apoenzyme. Creatinine to cytosine activity ratios varied from 1:3.75 (iron(II) chloride), to 1:0.9 (zinc chloride), to 1:0.06 (nickel chloride). For different preparations of the holoenzyme that ratio ranged from 1:0.45 to 1:1.10. Variable but significant quantities of zinc and iron were present in all preparations of the purified enzyme.     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.