Isolation of α and β Brain Tubulin Subunits after Alkaline Treatment of the Protein

We demonstrate here that brain purified tubulin can be dissociated into and subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, tubulin but not tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the tubulin was recovered in the detergent rich phase. The hydrophobic behavior observed for tubulin after its dissociation at pH 11.5 was not due to an irreversible change of the protein, because when the detergent rich phase containing tubulin was diluted with a buffer solution at pH 7.3 and the solution allowed to partition again, -tubulin is recovered in the aqueous phase. The detergent in the aqueous phase of the and tubulin preparations can be removed up to 90% by 12 h dialysis. The and subunits of tubulin from kidney and liver behave, in this phase separation system, like those of brain tubulin.     

Mammalian Sperm Tubulin: An Exceptionally Large Number of Variants Based on Several Posttranslational Modifications

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The - and -tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in and 0 to 15 in . Corresponding values for side-chain glutamyl residues were 0–6 in and 0–3 in . Additional variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in - and -tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.     

Alpha-tocopherol in the retinal outer segment of bovine eyes

Summary -Tocopherol was identified in lipid extracts of bovine retinal outer segment (ROS) preparations. Positive identification was obtained by the thin layer chromatographic characteristics of the tocopherol form and its oxidation product -tocopherylquinone, and by the ultraviolet spectrum of the oxidized and KBH₄-reduced form of the tocopherylquinone. In the ROS preparations used, -tocopherol chromanol was the predominant species, the quinone form accounting for 25% or less of the total. The concentration of -tocopherol in the ROS preparations was about 0.1 mole -tocopherol per mole rhodopsin, or about 1 nmole/mg, protein. Mitochondria from bovine retina contained about 0.4 nmole -tocopherol per mg protein.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

cDNA cloning of β-tubulin gene and organization of tubulin genes in Vigna radiata (mung bean) genome

We isolated an almost full-length cDNA clone containing -tubulin gene from a partial cDNA library of mung bean using chicken cDNA as probe. Cross-hybridization with chicken -tubulin cDNA and positive hybridization-selection and translation of mung bean mRNA established that this clone contains -tubulin sequences. We studied the organization of tubulin genes in mung bean. In this plant tubulin genes are organized in tandem repeats of alternating - and -tubulin genes. The 5.6 kb basis repeat unit which contains both - and -tubulin genes is repeated twenty times per haploid genome.Abbreviations SDS sodium dodecyl sulphate - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA, pH 7.4     

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.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Tyrosinated,detyrosinated and acetylated tubulin isotypes in rat brain membranes. Their proportions in comparison with those in cytosol

The heterogeneity of -tubulin and the relative proportions of the tubulin isotypes were investigated in brain membranes of rats of 1, 25 and 180 days of age by using four anti--tubulin antibodies: a) the monoclonal DM1A antibody, specific for -tubulin; b) the monoclonal 6-11B-1 antibody, specific for acetylated tubulin; c) a polyclonal antibody (Glu antibody), specific for detyrosinated tubulin; and d) a polyclonal antibody (Tyr antibody), specific for tyrosinated tubulin. We found that rat brain membranes contain the three tubulin isotypes mentioned above. The proportions of tyrosinated and detyrosinated tubulin relative to total -tubulin were somewhat lower in membrane than in cytosol in animals of 25 and 180 days of age. At day one of development, the proportions in membrane were similar to those found in cytosol. With respect to the acetylated form, it was about 20 times higher in membrane than in cytosol at the three ages studied. The proportion of acetylated tubulin was determined in different subcellular fractions: myelin, synaptic vesicles, mitochondria, microsomes, and plasma membrane. While the amount of total tubulin differed between the different subcellular fractions, the proportion of acetylated tubulin relative to total -tubulin was constant and similar to that found in total membranes. The proportion of acetylated tubulin was also investigated in non-neural tissues (kidney, liver and lung). Although values for cytosol were about 10-fold higher than that found in brain cytosol, no detectable values for membranes could be obtained in these organs.     

Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile

Biliverdin IX was purified from the bile of Atlantic salmon (Salmo salar) using a silica gel (Wakogel C-200) column. The yield was 49.5 mg per 100 ml of fresh bile and purity 95.3%. The biliverdin IX in the bile was quite stable when the bile was frozen at –80°C for a period of 40 days. However, 7.1% of the biliverdin IX was lost when the bile was stored at 4°C for 20 days. The purified biliverdin IX appeared as a single spot with R_f value of 0.25-0.27 on thin layer chromatography (TLC) and one main peak on high performance liquid chromatography (HPLC) at 436 or 650 nm. When the biliverdin IX was subjected to enzymic reduction with highly purified biliverdin reductase, two clear isobestic points were seen, at 384 and 670 nm. When the products of the reaction with biliverdin IX were extracted in butanol after completion of the reaction, one absorbance peak was observed at 468 nm. The time course of the reduction of biliverdin IX to bilirubin IX catalyzed by biliverdin reductase depended on reduced pyridine nucleotide. The time course of the NADPH-dependent reaction is different from that of the reaction with NADH. In the reduction of biliverdin IX , per mole of biliverdin IX reduced or per mole of bilirubin IX formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.     

Structural Features of Pregna-D′-pentaranes Determining Their Interaction with Three Rat Proteins

Competition of a number of progesterone 16,17-cycloalkane derivatives with ³H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16,17-cyclopropanoprogesterone, and 16,17-cyclopent-3-enoprogesterone and the selective ligands for serum pentaranophilin are 6-methyl-16,17-cyclohexanopregna-1,4-diene-3,20-dione and 3-hydroxy-16,17-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D we studied decreased the affinity of ligands for all the three proteins. The substitution of the ⁵-3-hydroxy grouping for the ⁴-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3,4-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found ,3- or ,6-linked to galactose (Gal), ,6-linked to N-acetylgalactosamine (GalNAc) or ,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in ,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by -galactoside ,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAc,6Gal1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-,6-lactosaminyl structure.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.     

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