The circulating α1-antitrypsin-elastase complex attacks the elastic lamina of blood vessels

Summary The aim of the study was to determine the destination of the ₁-antitrypsin-elastase complex, which is found in circulating blood after the peroral administration of elastase. The complex was made in vitro by mixing hog pancreatic elastase with human ₁-antitrypsin and then injected intravenously into rats and mice. Tissues taken at various times after injection were subjected to histochemical staining using an antibody against elastase. Light micro-scope observations revealed dense deposition of reaction products in the elastic lamina of the arterioles; moderate or slight deposits were seen in the tissues surrounding arteries, in the tubular epithelial cells of the proximal convoluted tubules in the kidney, and in the pancreatic ducts.Immuno-electron microscopy revealed heavy deposition of the reaction product in the elastic lamina of the small arteries and arterioles; some dissolution of the elastic fibers was also evident. Pinocytic uptake of the ₁-antitrypsin-elastase complex was observed on the abluminal surface of endothelial cells and in smooth-muscle cells bordering the elastic lamina of arterioles. The endothelial cells of the arteries and arterioles retained their normal morphological appearance, although local desquamation was observed in some animals. The results indicate that, when the ₁-antitrypsin-elastase complex is present in the circulating blood, it is incorporated into the elastic lamina through the endothelial layer. This results in liquefaction of the lamina, desquamation of endothelial cells and leakage of the complex into the perivascular tissues via the vascular walls. However, some of the complex seems to be excreted very quickly from the kidneys.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

The stereospecificity of α-chymotrypsin

1. The rates of deacylation of acyl-alpha-chymotrypsins in which the hydrogen-bonding capacity of the acylamino group of the substrate has been systematically removed were measured. 2. The ratio of deacylation rates of l- and d-acyl-enzymes is found to depend largely on the existence in the substrate of an amido -NH- group. 3. The data presented agree with the postulate that the stereospecificity of alpha-chymotrypsin is exercised in catalytic rather than binding steps, and that the active site of the enzyme presents three loci to the substrate: the site containing the catalytic functionalities (including serine-195), the hydrophobic area for amino acid side-chain binding, and a hydrogen-bond acceptor site for acylamino group binding. 4. It is noted that, though the hydrogen-bonding site is crucial for the stereospecificity, the free energy of binding of substrates and inhibitors is dominated by the hydrophobic interaction. 5. It is tentatively proposed that alpha-chymotrypsin selects a high-energy conformation of the substrate when the latter binds at the enzyme's active site.     

The polymerization of α-aminopropionitrile

The mixture of -aminopropionitrile and ,-iminodipropionitrile polymerized to solidify almost at the temperature near 0°C during 8 years. The conversions based on decreasing of those reactants were 61 and 98% at 4 and 8 years, respectively. The fractionation of 4 and 8 years product using Sephadex G 10 yielded their predominant amounts in the oligomer and polymer section, respectively. The oligomer section product of 8 years product was analyzed by means of ion exchange chromatography and its trimethylsilyl derivative was also analyzed by means of gas chromatography combined with mass spectrometry. These results identified dialanine and trialanine and their amides and nitriles.     

Molecular dynamics study of an α-cyclodextrin-phosphatidylinositol inclusion complex

Cyclodextrins are cyclic oligosaccharides known for their ability to include substrate molecules in their hydrophobic cavity. Moreover, cyclodextrins show a hemolytic activity when mm concentrations are added to blood. This hemolysis is commonly interpreted as a massive dissociation of phospholipids from the cell membrane due to the formation of complexes with the cyclodextrins. In the literature, a complexation between -cyclodextrin ( CD) and phosphatidylinositol (PI) specific to the inositol headgroup has been proposed. But the need for the detailed interaction mechanism between the two molecules motivated the present work based on molecular dynamics simulations. Investigation of long range electrostatic interactions shows that a mutual approach of the molecules is only possible when the primary hydroxyl side of CD faces the inositol headgroup of PI. This orientation is also the most favourable from adiabatic- and free-energy profiles calculated along a reaction coordinate that leads to an inclusion of PI into a CD. For free energy simulations, partial hydration of the model has been used. A study of glycosidic bond dihedral angles in CD shows an increase in dihedral fluctuations before complexation and a dihedral freezing once the complex is formed.     

The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.     

The Synthesis of Dilnethyl α-Sorigenin

1,6,8-Trimethoxy-3-hydroxymethyl-2-naphthoic acid lactone (IV) was synthesized from benzoic acid in 21 steps. This lactone (IV) was completely identical with authentic dimethyl α-sorigenin, obtained by the methylation of natural α-sorigenin. Herewith the structure of α-sorigenin was confirmed to be 1,8-dihydroxy-6-methoxy-3-hydroxymethyl-2-naphthoic acid lactone (III).     

The Synthesis of α-Methylamino Acids

α-Methyltryptophan, α-methylhistidine, and α-methyldopa were prepared by the reaction of α-isocyanopropionates with gramine methiodide, the acetoxymethylimidazole derivative, and protected 3,4-dihydroxybenzyl bromides, respectively.     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

Carbohydrate recognition by an architecturally complex α-N-acetylglucosaminidase from Clostridium perfringens

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-D-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-D-glucosamine-α-1,4-D-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-D-glucosamine-α-1,4-D-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Genomic rearrangement of the α-globin gene complex during mammalian evolution

In man, the gene for hydroxyacyl glutathione hydrolase (HAGH; glyoxalase II) is closely linked to the α-globin locus (HBα) on Chromosome 16. HAGH polymorphism in the mouse has now enabled the mapping of the murine homologue. Deletion mapping, congenic strain studies, and characterization of 41 recombinant inbred strains establish that the mouseHagh locus lies very close to the α-globin pseudogene (Hba-ps4) in the vicinity of the major histocompatibility locus (H-2) on chromosome 17. Several other loci have been identified previously that are also closely linked to the human α-globin locus but near the α-globin pseudogeneHba-ps4 in the mouse. These linkage relationships suggest that during the evolution of mice a translocation occurred that subdivided the α-globin locus, leaving one inactive α-globin gene still associated with theHagh locus and linked sequences, while moving and inserting the active α-globin locus and all distal sequences into an internal location on another autosome, the predecessor to mouse chromosome 11.     

The structure of the α-keratin microfibril

Quantitative measurements of the intensity of the meridional reflections in the X-ray-diffraction pattern of alpha-keratin are shown to be consistent with a microfibril structure in which a surface lattice with an axially projected period around 200 A is subject to a periodic interruption with an axially projected period of 470 A. Taken in conjunction with recent evidence on the chemical structure of alpha-keratin and other intermediate filaments this finding enables an elaboration to be made of a model proposed earlier by RDB Fraser, TP MacRae, & E Suzuki (J. Mol. Biol. 108, 435-452, 1976) for the alpha-helical framework of the microfibril. The disposition and connectivity of the helical segments suggested here provides a straightforward explanation of a number of recent physicochemical and electron-microscopical observations on intermediate filaments and provides a starting point for the development of models for the framework of other intermediate filaments.     

The NAD-linked α-glycerophosphate dehydrogenase of trypanosomes

NAD-linked α-glycerophosphate dehydrogenase plays a key role in the α-glycerophosphate cycle of $\text{Trypanosoma brucei}$. The activity in cell lysates was ample for this role. The enzyme was activated by salts (e.g. MgCl₂ or NaCl); it had a broad pH-optimum for the reduction of dihydroxyacetone phosphate centred at pH 7.4, with an apparent K_m of 0.5 mM; and it was weakly bound to particulate components of cell lysates. The enzyme from $\text{T. vivax}$ was similar to that of $\text{T. brucei}$. These trypanosomal enzymes resemble that of the trypanosomatid $\text{Crithidia fasciculata}$, but are rather different from the enzymes of mammals, birds and insects.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Genomic organization of the complex α-gliadin gene loci in wheat

To better understand the molecular evolution of the large -gliadin gene family, a half-million bacterial artificial chromosome (BAC) library clones from tetraploid durum wheat, Triticum turgidum ssp. durum (2n=4x=28, genome AB), were screened for large genomic segments carrying the -gliadin genes of the Gli-2 loci on the group 6 homoeologous chromosomes. The resulting 220 positive BAC clones—each containing between one and four copies of -gliadin sequences—were fingerprinted for contig assembly to produce contiguous chromosomal regions covering the Gli-2 loci. While contigs consisting of as many as 21 BAC clones and containing up to 17 -gliadin genes were formed, many BAC clones remained as singletons. The accuracy of the order of BAC clones in the contigs was verified by Southern hybridization analysis of the BAC fingerprints using an -gliadin probe. These results indicate that -gliadin genes are not evenly dispersed in the Gli-2 locus regions. Hybridization of these BACs with probes for long terminal repeat retrotransposons was used to determine the abundance and distribution of repetitive DNA in this region. Sequencing of BAC ends indicated that 70% of the sequences were significantly similar to different classes of retrotransposons, suggesting that these elements are abundant in this region. Several mechanisms underlying the dynamic evolution of the Gli-2 loci are discussed.     

The α-glucuronidase-encoding gene of Trichoderma reesei

The Trichoderma reesei cDNA coding for α-glucuronidase (GLRI), which releases glucuronic acid attached to xylose units of xylan, was cloned and sequenced. The deduced N-terminal amino acid (aa) sequence of the protein was verified by sequencing of the purified GLRI. The aa sequence of the GLRI displayed no similarity with any aa sequence available in the data bases.     

The binding of inhibitors to α-chymotrypsin

1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.