Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new -tubulin isoform from sunflower we named -tubulin. -tubulin is the most divergent higher-plant -tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that -tubulin expression is restricted to the male gametophyte. -tubulin mRNA represents 90% of -tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, -tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that -tubulin is specific to this family. Our results suggest that -tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that -tubulin is found in a complex with -tubulin in mature sunflower pollen.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

Convenient and simple homologation of Nα-urethane protected α-amino acids to their β-homologues with concomitant pentafluorophenyl ester formation

The Wolff rearrangement of -aminodiazoketonesderived from N-urethane protected -amino acids in the presence of pentafluorophenol catalyzed by silver acetate at low temperature results in the homologation of Fmoc-/Boc-/Z--amino acids to -amino acids withconcomitant formation of the corresponding pentafluorophenylesters in good yield and purity.     

Interleukin-6 Induced Suppression of Bovine Parathyroid Hormone Secretion

Calcium disturbances in the critically ill coincide with elevations of proinflammatory cytokines. The effects of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) on parathyroid hormone (PTH) secretion were investigated. IL-6 and TNF- had no acute effect on PTH secretion in extracellular Ca²⁺ concentrations of 0.5, 1.25 and 3.0 mM. In contrast to TNF-, cultures for 24 h in the presence of l0 ng/mL of IL-6 showed decreased PTH secretion by 51% and 29% in 0.5 mM and 1.25 mMCa²⁺ respectively. Neither IL-6 nor TNF-, affected cytoplasmic Ca²⁺ of the cells. We conclude that PTH secretion in vitro can be suppressed by IL-6 at clinically relevant concentrations. This suppression may aggravate hypocalcemia of the critically ill and attenuate the conventionally strong stimulation of the PTH release by reduction in serum calcium.     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Primary structure of hemoglobin α-chain ofColumba livia (gray wild pigeon)

Primary structure of hemoglobin of -chain ofColumba livia is presented. The separation of -chain was obtained from globin by ion-exchange chromatography (CMC-52) and reversed-phase HPLC (RP-2 column). Amino acid sequence of intact as well as tryptic digested chain was determined on gas-phase sequencer. Structure is aligned homologously with 21 other species. Among different exchanges, positions 24 (TyrLeu), 26 (AlaGly), 32 (MetLeu), 64 (AspGlu), 113 (LeuPhe), and 129 (LeuVal) are unique to pigeon hemoglobin. The various exchanges in -chain are discussed with reference to evolution and phylogeny. The results show that the order Columbiformes is evolutionarily closer to the order Anseriformes. Since the pigeon is homogeneous, having HbA (^A-chain) and lacks ^D-chain, its phylogenetic placement could be established among birds having single hemoglobin components.     

Kinetic study of the cytotoxic effect of α-sarcin,a ribosome inactivating protein fromAspergillus giganteus,on tumour cell lines: protein biosynthesis inhibition and cell binding

-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. -Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by -sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with -sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by -sarcin reveals an -sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external -sarcin concentration. A saturable component for the action of -sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of -sarcin.