Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Characterization of a β-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Treatment by EDTA of purified plasma membranes from MF₂S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ to ouabain. When added back with Ca²⁺ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca²⁺ it was able to restore the original sensitivity to ouabain of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca²⁺ the same quantitative biological activity on ouabain sensitivity of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. A possible interaction between β-actinin, calmodulin and membrane-bound $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is discussed.     

Metabolism of Δ4-cis-PGF1α in the monkey

This isomer of PGF_2α is relatively resistant to metabolic degradation in the Cynomolgus monkey. Thus, 16–20 per cent of the amount injected was excreted unchanged in the urine. Five metabolites with 20, 18, 16 and 14 carbon atoms in the skeleton were identified. The data are similar to those earlier seen in the rat and further support the idea that this analogue of PGF_2α could have a long half-life time in the mammalian body and thus a long duration of its pharmacological actions.     

Determination of H+e− ratios in chloroplasts with flashing light

Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H₂O₂ formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratios of 3 and 4 for ferricyanide and methyl viologen, respectively.In continuous light of low intensity, we obtained the same $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio approached 2 as a limit for both acceptors.A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a ≈ 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6–10 ms.     

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal,testis and ovary during late gestation

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ (3β-HSDH) was measured in the rhesus monkey ($\text{Macaca mulatta}$) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [³H]-pregnenolone to [³H]progesterone. The maximum enzyme velocity (V_m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg^?1protein) than in microsomes from the other tissues. Testicular V_m was greater than either ovarian or placental V_m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg^?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.     

Comparison of red cell and kidney (Na+ + K+)-ATPase at 0°C

Human red cell and guinea pig kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ were phosphorylated at 0°C. Using concentrations of ATP ranging from 10^?6 to 10^?8 M, ATP-dependent regulation of reactivity is observed with red cell but not kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C. In particular, with the red cell enzyme only, the following are observed: (i) the ratio of enzyme-bound ATP (E·ATP, measured by the pulse-chase method of Post, R.L., Kume, S., Tobin, T., Orcutt, B. and Sen, A.K. (1969) J. Gen. Physiol. 54, 306s-326s) to steady-state level of total phosphoenzyme (EP) decreases with decrease in ATP concentration and (ii) the apparent turnover of phosphoenzyme (ratio of Na⁺-stimulated ATP hydrolysis to level of total EP at steady state) also varies as a function of ATP concentration. In addition, when EP is formed at very low ATP (0.02 μM), and then EDTA is added, rapid disappearance of a fraction of EP occurs, presumably due to ATP resynthesis, only with the red cell enzyme. These differences in behaviour of the red cell and kidney enzymes are explained on the basis of the observed predominance of K⁺-insensitive EP in red cell, but K⁺-sensitive EP in kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C.     

Net CI− flux in short-circuited skin of Rana pipiens: Ouabain sensitivity and Na+ + K+ dependence

Skin of Rana pipiens, like many other species, is considered to actively trassport only Na⁺ when bathed in Ringer's solution on both sides. However, net CI^? influx was previously described by us in short-circuited skin of Rana pipiens, in the summer season. The skins were of low PD (5–30 mV) and high CI^? conductance. Comparison of these findings with other series indicated inverse seasonal variation between PD and CI^? conductance. It was postulated that active CI^? transport exists at all CI^?-conductance levels but at higher PD is too small to be be easily detected. This report evaluates CI^? transport across skins of higher PD (and lower conductance) in winter and further characterizes the system. Net CI^? influx was demonstrated over a wide PD range under short-circuit conditions and was inversely related to the magnitude of the open-circuit PD. It was inhibited by ouabain and dependent upon Na⁺ in the outside medium and K⁺ in the inside medium. It is concluded that this is the same system present in skin of Leptodactylus ocellatus and may not be unique to these two species. It appears to be distinctly different from the acetazolamide-sensitive influx system seen in “low-CI^?” solutions in skins of numerous species. The findings are discussed in terms of (1) parallel Na⁺ and CI^? active transport mechanisms and (2) a neutral NaCI-pump model, recently proposed by Rehm.     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

Cytotoxicity of a hybrid prepared by coupling diphtheria toxin A-chain with immunoglobulin Fab′ with N,N′-o-phenylenedimaleimide

A hybrid protein was prepared by coupling the A-chain of diphtheria toxin with the Fab′ fragment of immunoglobulin with $\text{N}\text{,}\text{N′}\text{-}\text{o}$-phenylenedimaleimide (PDM). Although in this hybrid, the two components were linked with each other with bonds which could not be reductively cleaved with 2-mercaptoethanol as in a hybrid cross-linked with a disulfide bond (e.g. Fab′-S-S-A-chain), it exhibited a potent cytotoxicity $\text{in}\text{vitro}$, one-third of that of Fab′-S-S-A-chain, against the target L1210 cells.     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

Purification and characterization of a glycoprotein,from normal human liver,which has the same molecular weight and chemical properties as plasma α1-antrypsin

An $\text{α}{}_1\text{-}\text{mantitrypsin-like}$ material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma $\text{α}{}_1\text{-}\text{antrypsin}$. The liver material, in contrast to a previous report¹ has the same molecular weight as the corresponding normal plasma $\text{α}{}_1\text{-}\text{antrypsin}$. The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma $\text{α}{}_1\text{-}\text{antrypsin}$. Amino acid and carbohydrate compositions of the liver material are similar to those of $\text{α}{}_1\text{-}\text{antrypsin}$ obtained from the plasma. The $\text{α}{}_1\text{-}\text{antrypsin-like}$ material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy.     

Effects of NO2? and NO3? on the Fe(III)EDTA reduction in a chemical absorption–biological reduction integrated NOx removal system

The biological reduction of Fe(III) ethylenediaminetetraacetic acid (EDTA) is a key step for NO removal in a chemical absorption–biological reduction integrated process. Since typical flue gas contain oxygen, NO₂ ⁻ and NO₃ ⁻ would be present in the absorption solution after NO absorption. In this paper, the interaction of NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA reduction was investigated. The experimental results indicate that the Fe(III)EDTA reduction rate decrease with the increase of NO₂ ⁻ or NO₃ ⁻ addition. In the presence of 10 mM NO₂ ⁻ or NO₃ ⁻, the average reduction rate of Fe(III)EDTA during the first 6-h reaction was 0.076 and 0.17 mM h⁻¹, respectively, compared with 1.07 mM h⁻¹ in the absence of NO₂ ⁻ and NO₃ ⁻. Fe(III)EDTA and either NO₂ ⁻ or NO₃ ⁻ reduction occurred simultaneously. Interestingly, the reduction rate of NO₂ ⁻ or NO₃ ⁻ was enhanced in presence of Fe(III)EDTA. The inhibition patterns observed during the effect of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction experiments suggest that Escherichia coli can utilize NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA as terminal electron acceptors.     

The Mrp system: a giant among monovalent cation/proton antiporters?

Mrp systems are a novel and broadly distributed type of monovalent cation/proton antiporter of bacteria and archaea. Monovalent cation/proton antiporters are membrane transport proteins that catalyze efflux of cytoplasmic sodium, potassium or lithium ions in exchange for external hydrogen ions (protons). Other known monovalent cation antiporters are single gene products, whereas Mrp systems have been proposed to function as hetero-oligomers. A mrp operon typically has six or seven genes encoding hydrophobic proteins all of which are required for optimal Mrp-dependent sodium-resistance. There is little sequence similarity of Mrp proteins to other antiporters but three of these proteins have significant sequence similarity to membrane embedded subunits of ion-translocating electron transport complexes. Mrp antiporters have essential roles in the physiology of alkaliphilic and neutralophilic Bacillus species, nitrogen-fixing Sinorhizobium meliloti and in the pathogen Staphylococcus aureus, although these bacteria contain multiple monovalent cation/proton antiporters. The wide distribution of Mrp systems leads to the anticipation of important roles in an even wider variety of pathogens, extremophiles and environmentally important organisms. Here, the distribution, established physiological roles and catalytic activities of Mrp systems are reviewed, hypotheses regarding their complexity are discussed and major open questions about their function are highlighted.     

The PINK1/Parkin pathway: a mitochondrial quality control system?

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria.     

Photoconvertible protochlorophyll(ide)635650 in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.