α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins

Clusters of di-, tri-, and tetra-antennary -D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide {Man (16)[Man (13)]Man} haptens to poly-isocyanate or -isothiocyanate tethering cores. The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively. Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses. In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl -D-mannopyranoside against pea lectin and Con A, respectively. Compounds 30 and 31 were thus 35 and 289 fold more potent than the reference monosaccharide based on their hapten contents. As a general observation, the ligands bearing the Man (16)[Man (13)]Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands.     

Arabidopsis thaliana ‘extra-large GTP-binding protein’ (AtXLG1): a new class of G-protein

Heterotrimeric GTP-binding proteins, composed of , , and subunits, are involved in signal transduction pathways in animal and plant systems. In plants, physiological analyses implicate heterotrimeric G-proteins in ion channel regulation, light signaling, and hormone and pathogen responses. However, only one class of plant G genes has been identified to date. We have cloned a novel gene, Arabidopsis thaliana extra-large GTP-binding protein (AtXLG1). AtXLG1 appears to be a member of a small gene family and is transcribed in all tissues assayed: roots, leaves, stems, flowers, and fruits. The conceptually translated protein from AtXLG1 is 99 kDa, twice as large as typical G proteins. The carboxy-terminal half of the AtXLG1 protein has significant homology to animal and plant G proteins. This region includes a GTP-binding domain, a predicted helical domain, and an aspartate/glutamate-rich loop, which are characteristics of G's. Despite the absence of some of the amino acids implicated in GTP binding and hydrolysis by crystallographic and mutational analyses of mammalian G's, recombinant AtXLGl binds GTP with specificity. The amino-terminal region of AtXLGl contains domains homologous to the bacterial TonB-box, which is involved in energy transduction between the inner and outer bacterial membranes, and to zinc-finger proteins. Given the unique structure of AtXLG1, it will be of interest to uncover its physiological functions.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

Archaeal elongation factor 1α from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> interacts with the eubacterial antibiotic GE2270A

The thiazolyl-peptide antibiotic GE2270A, an inhibitor of the elongation factor Tu from Escherichia coli (EcEF-Tu), was used to study the effects produced in the biochemical properties of the archaeal functional analogue elongation factor 1 from Sulfolobus solfataricus (SsEF-1). GE2270A did not substantially affect the poly(U)-directed-poly(Phe) incorporation catalyzed by SsEF-1 and the formation of the ternary complex SsEF-1·GTP·Phe-tRNA^Phe. On the other hand, the antibiotic was able to increase the GDP/GTP exchange rate of SsEF-1; nevertheless, this improvement was not associated with an increase in the catalytic activity of the enzyme. In fact, GE2270A inhibited both the intrinsic GTPase of SsEF-1 (GTPase^Na) and that stimulated by ribosomes. Interestingly, GTPase^Na of both intact and C-terminal-deleted SsEF-1 resulted in a greater sensitivity to the antibiotic with respect to SsEF-1 lacking both the M- and C-terminal domains. This result suggested that, similar to what is found for EcEF-Tu, the M domain of SsEF-1 is the region of the enzyme most responsible for the interaction with GE2270A. The different behavior observed in the inhibition of protein synthesis with respect to EcEF-Tu can be ascribed to the different adaptive structural changes that have occurred in SsEF-1 during evolution.     

High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions

Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.¹²⁵I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the ₂₂ heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the ₂₂ association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP adenosine 5-triphosphate - DTT dithiothreitol - SDS sodium dodecyl sulfate - DSS disuccinimidyl suberate - NEM N-ethylmaleimide - IGF-I insulin-like growth factor-I - EDTA ethylenediaminetetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon 2b (IFN2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN2b were analysed by [³H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Clinical and immunological evaluation of 20 patients with advanced colorectal cancer treated with high dose recombinant leukocyte interferon-αA (rIFNαA)

Summary A total of 20 patients with advanced colorectal cancer received recombinant leukocyte interferon-A (rIFNA) either chronically (group I: twice a week up to 20×10⁶ IU/m² i.m.) or cyclically (group II: 1–4 periods of 8 consecutive days up to 20×10⁶ IU/m² i.m. daily at 20-days intervals) over a period of 12 weeks. There was 1 partial response, 1 mixed response and 1 patient with stable disease, whilst 17 patients had progressive disease. Median survival was 15.5 months. Survival was significantly shorter when the extent of hepatic disease was >25% (P=0.05), extrahepatic disease was extensive (P<0.005), alkaline phosphatase level was >2× normal (P<0.02), or performance status was <100% (P<0.001). Toxicity consisting mainly of fever, fatigue, anorexia and weight loss was serious in group I and minimal in group II. Administration of rIFNA led to a short lived augmentation of natural killer (NK) cell activity. In the cyclically treated group this was a recurrent phenomenon whereas a marked lasting depression of NK cell activity was seen in chronically treated patients. Interferon- production capacity was significantly stimulated during rIFNA therapy. The differences in toxicity and immunostimulatory effects between the two schedules may be of importance in the design of further studies.This trial was supported in part by Hoffmann-La Roche, Basle     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

The molecular basis of HbH disease in Taiwan

Summary We have determined the molecular characteristics of -thalassemia in 12 HbH subjects from Taiwan by restriction endonuclease mapping with -and -specific probes. We have found four types of defects in the -thalassemia-2 genetic determinant: ^3.7 type I; ^4.2; ^CS; and ^T. All HbH subjects carried the ——^SEA genotype in the -thalassemia-1 determinant. At least two different subtypes of ——^SEA genotype were observed in this study.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Alpha-thalassemia in Papua New Guinea

Summary A study of the distribution of -thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for -thalassemia 2 and -thalassemia 1 caused respectively by either deletion of one or both of the duplicated -globin genes. -Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of -thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with -thalassemia and -thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single -globin gene deletion revealed a predominance of the –^4.2 type in general, except in some regions in the south where the –^3.7 type is prevalent. The –^3.7 I subtype is the common form of the –^3.7 deletion in the PNG mainland. The –^3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.     

Double-immunofluorescent staining of isolated smooth muscle cells

Summary Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying -actinin from chicken gizzards was used to provide antigen for raising anti--actinin. Fluorescein isothiocyanate-labelled anti--actinin (FAA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlony gels against myosin, tropomyosin, actin, and -actinin showed that antimyosin reacted only with myosin, anti--actinin only with -actinin. Anti--actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of -actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of -actinin much easier to interpret.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.