Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring

Summary [¹³C₅]-2-Deoxy-d-ribose, synthesized from [¹³C₆]-d-glucose (98% ¹³C), was coupled with thymine to give [1,2,3,4,5-¹³C₅]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3-phosphoramidite derivative and was then incorporated into a dodecamer 5-d(CGCGAATTCGCG)-3 by solid-phase DNA synthesis. Preparation of 0.24 mole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the ¹³C labels, all of the ¹H-¹H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Beta-adrenergic control of blood oxygen affinity in acutely hypoxia exposed rainbow trout

Summary Acute exposure of rainbow trout to hypoxic water ( =40 mmHg, 15 °C) caused a significant (P<0.01) increase in blood O₂ affinity, from the normoxicP ₅₀ value (at pH_e 7.93) of 23.2±1.1 mmHg to about 19 mmHg, within 5 min. Specimens injected with the -antagonist propranolol showed no change in bloodP ₅₀, despite a more pronounced reduction of arterial during the hypoxic exposure.The change in bloodP ₅₀ coincided with an increase in plasma catecholamines, notably noradrenaline. There was no change in the molar ratios of ATPHb₄ and GTPHb₄. The altered bloodP ₅₀, however, correlated with an alkalinization and an increased sodium concentration of the red cells. This red cell alkalinization can be explained by -adrenergic stimulation of a membrane bound Na⁺/H⁺ antiporter.Propranolol injection into normoxic resting trout caused a significant decrease in and increase in indicating -adrenergic control of gas exchange in the gills.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Gross nitrogen transformations in soils from uncut and cut boreal upland and peatland coniferous forest stands

Gross and net nitrogen (N) ammonification and nitrification were measured in soils from an uncut and recently cut upland and peatland conifer stand in northwestern Ontario, Canada. Rates of gross total inorganic N immobilization were similar to gross mineralization, resulting in low net mineralization rates in soils from all four upland and peatland conifer stands. Gross ammonification rates were variable but similar in soils from uncut and cut peatland hollows (18–19mgNkg^–1day^–1) and upland forest floor soils (14–19mgNkg^–1day^–1). Gross ammonium ( ) immobilization rates were also variable but similar to ammonification rates. Median gross nitrification rates were within 0–2mgNkg^–1day^–1 in soils from all four upland and peatland cut and uncut stands, although rates were consistently higher for the soils from the cut stands. Large variability in gross nitrification rates were observed in peatland soils, however the highest gross nitrification rates were measured in saturated peatland soils. Net rates remained low in the soils from all four stands due to high nitrate ( ) immobilization and very fast turnover (<0.2 day). Our results suggest that potential losses may be negated by high immobilization in uncut and cut boreal forest stands. This study reveals the potential for the interaction of N production and consumption processes in regulating N retention in upland and peatland conifer forests, and the resilience of the boreal forest to disturbance.     

Beta-Adrenergic modulation of K+ current in human T lymphocytes

Summary The whole-cell voltage-clamp technique was employed to study the -adrenergic modulation of voltage-gated K⁺ currents in CD8⁺ human peripheral blood lymphocytes. The -receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K⁺ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the -blocker propranolol. Bath application of dibutyryl cAMP (1mm) mimicked the effects of isoproterenol on both K⁺ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI_5–24 (2–5 m), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K⁺ currents in response to isoproterenol was mimicked by the internal application of GTP--S (300 m) and by exposure of the cell to cholera toxin (1 g/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K⁺ conductance in T lymphocytes can be modulated by -adrenergic stimulation. The effects of -agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K⁺ current has been found to decrease the proliferative response in T lymphocytes, the -adrenergic modulation of K⁺ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Mg2+ Induces Conformational Changes in the Catalytic Subunit of Phosphorylase Kinase, Whether by Itself or as Part of the Holoenzyme Complex

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition ()₄. Previous studies have revealed that the activity of its catalytic subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb 79) has been generated against isolated subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the -calmodulin binary complex, and the free subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb 79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the subunit containing the epitope for mAb 79. The activating ligand Mg²⁺ also stimulated the binding of the PhK holoenzyme to immobilized mAb 79, as well as the binding of mAb 79 to immobilized subunit. Thus, Mg²⁺ increases the accessibility of the mAb 79 epitope in both the isolated subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg²⁺ on the quaternary structure of the PhK holoenzyme are directly mediated by the subunit.     

Characteristics of the Inhibitory Effect of Calmodulin on Specific [125I]Omega-Conotoxin GVIA Binding to Crude Membranes from Chick Brain

The characteristics of the inhibitory effect of calcium ion (Ca²⁺)/calmodulin (CaM) on specific [¹²⁵I]-omega-conotoxin GVIA (¹²⁵I--CTX) binding and on the labeling of ¹²⁵I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca²⁺/CaM depended on the concentrations of free Ca²⁺ and CaM. The IC₅₀ values for free Ca²⁺ and CaM were about 2.0 × 10^–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca²⁺/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca²⁺ channel ₁ subunits) with ¹²⁵I--CTX using a cross-linker. These results suggest that Ca²⁺/CaM affects specific ¹²⁵I--CTX binding sites, probably N-type Ca²⁺ channel ₁ subunits, in crude membranes from chick whole brain.     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Antitumor effect of recombinant tumor necrosis factor-α against murine sarcomas at visceral sites: tumor size influences the response to therapy

Summary We examined the antitumor efficacy of rTNF- administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (>100 foci of 0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF- (5–10 g, single dose i. v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s. c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF- failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (>100 foci of 0.5 mm diameter) hepatic metastases at single i. v. doses up to 20 g, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s. c. with MCA-106 sarcoma and treated with rTNF- as a single i. v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF- injection, when the tumor had achieved a size of 5–6 mm in diameter. Since tumor size appeared important for rTNF- susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5–6 mm in diameter. Increasing doses of rTNF- (up to 8 g) given as a single i. v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF- administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF- susceptibility not only for tumors growing at s. c. sites but also for those established at visceral sites.Howard Hughes Medical Institute Research Scholar Abbreviations used: rTNF-, recombinant tumor necrosis factor-; B6 mice, C57BL/6 mice; MCA, 3-methylcholanthrene; HBSS, Hanks' balanced salt solution; LAK, lymphokine-activated killer; rIL-2, recombinant interleukin-2     

Screening for naturally occurring apolipoprotein A-I variants: apo A-I(ΔK107) is associated with low HDL-cholesterol levels in men but not in women

Isoelectric focussing (IEF) in carrier ampholyte-generated pH gradients and hybrid isoelectric focussing (HIEF) in immobilized pH gradients under nondenaturing conditions were used in parallel to screen 5,500 plasma samples for naturally occurring variants of apolipoprotein A-I (apo A-I). The following defects were identified in four unrelated subjects heterozygous for apo A-I variants: apo A-I(K107)(2 ×), apo A-I(K107M)(1 ×), and apo A-I(E41R)(1 ×). The later variant is a novel finding. Family studies did not reveal any association of apo A-I(K107M) and apo A-I(E41R) with dyslipidemia, but identified several heterozygotes for apo A-I(K107) who had low levels of high density lipoprotein (HDL)cholesterol. Therefore, and since the apo A-I(K107) is the most frequent apo A-I variant in Germany (1 5,000) we evaluated our data and that reported from 11 families with 32 heterozygous carriers and 30 unaffected controls. This analysis revealed that apo A-I(K107) is associated with lower HDL-cholesterol (-30%) and higher triglycerides (+ 48%) in men but not in women as compared with unaffected family members as well as with controls from the Prospective Cardiovascular Münster (PROCAM) study. Moreover, 11 of 15 male apo AI(K107) heterozygotes but only 2 of 17 female apo AI(K107) heterozygotes had HDL-cholesterol levels below the 20th percentile of sex-matched controls from the PROCAM study. We conclude that heterozygosity for apo A-I(K107) decreases HDL-cholesterol and increases triglycerides in men but not in women.     

Synthesis of oligoguanylates on oligocytidylate templates

Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb²⁺ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb²⁺-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn²⁺, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G Guanosine - Gp guanosine 2(3)-phosphate - pG guanosine 5-phosphate - Gp! guanosine cyclic 2,3-phosphate - ImpG guanosine 5-phosphorimidazolide - ImpG^* [8-¹⁴C]-guanosine 5-phosphorimidazolide - pGp 5-phosphoguanosine 2(3)-phosphate - G²pG guanylyl-[2-5]-guanosine - G³pG guanylyl-[3-5]-guanosine - ImpGpG 5-phosphorimidazolide of GpG - (pG)_n (n = 2,3) oligomers of pG - GppG P₁, P₂-diguanosine 5-diphosphate - GppGpG 5-[guanosine 5-pyrophosphate] of GpG - NH₂pG guanosine 5-phosphoramidate - (pG)₄₊ tetramer and higher oligoguanylates with 5 terminal phosphate - oligo(G) oligoguanylate - Cp cytidine 2(3)-phosphate - Cp! cytidine cyclic 2,3-phosphate - (Cp)_n–1 Cp! (n= 2,3,4) oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates - oligo(C) oligocytidylate - poly(C) polycytidylic acid - poly(U) polyuridylic acid - poly(C,G) random copolymer of C and G - BAP bacterial alkaline phosphatase (E. coli) - EDTA ethylenediaminetetraacetic acid - R_f chromatographic mobility     

Correlation between 15N NMR chemical shifts in proteins and secondary structure

Summary An empirical correlation between the peptide ¹⁵N chemical shift, ¹⁵N_i, and the backbone torsion angles _i, _i–1 is reported. By using two-dimensional shielding surfaces (_i1–1), it is possible in many cases to make reasonably accurate predictions of ¹⁵N chemical shifts for a given structure. On average, the rms error between experiment and prediction is about 3.5 ppm. Results for threonine, valine and isoleucine are worse (4.8 ppm), due presumably to ₁-distribution/-gauche effects. The rms errors for the other amino acids are 3 ppm, for a typical maximal chemical shift range of 15–20 ppm. Thus, there is a significant correlation between ¹⁵N chemical shift and secondary structure.     

High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium

A mixture of dihexanoyl phosphatidylcholine and dimyristoylphosphatidylcholine in water forms disc-shaped particles, often referred toas bicelles [Sanders and Schwonek (1992) Biochemistry, 31, 8898–8905].These adopt an ordered, liquid crystalline phase, which can be maintained atvery low concentrations of the bicelles (down to 3% w/v). At thisconcentration the spacing between individual bicelles, on average, exceeds300 Å. The bicelles are shown to have a negligible effect on therotational diffusion of ubiquitin as judged by the ¹⁵NT₁ values of the backbone amides relative to those inisotropic aqueous solution. The protein exhibits a residual degree ofalignment which is proportional to the bicelle concentration, andapproximately collinear with ubiquitin's rotational diffusion tensor. Thedegree of alignment obtained offers unique opportunities for studying theprotein's structure and dynamics.