Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

Microtubule-associated protein MAP2 preferentially binds to a dA/dT sequence present in mouse satellite DNA

Microtubule-associated protein MAP2 binds to the Sau96.1 restriction monomer fragment of mouse satellite DNA. This fragment is also present in a lower proportion in bulk DNA. The digestion of MAP2-Sau96.1 fragment complex by DNase results in the protection of certain nucleotide sequences. The sequence poly(dA)4/poly(dT)4 is mainly protected against DNase digestion.     

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.     

Microinjection of human cell extracts corrects xeroderma pigmentosum defect

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.     

Reinvestigation of the shape and state of hydration of the skeletal myosin subfragment 1 monomer in solution

Hydrodynamic calculations lead to the conclusion that chymotryptic (or ethylenediaminetetraacetic acid) myosin S1 in solution (hydrated), at 1-5 degrees C, can be modeled as a prolate ellipsoid, with an axial ratio lying between p = 1.0 and 2.5 (major axis between 100.5 A, for p = 1.0, and 162.5 A, for p = 2.5). The degree of hydration is considerable (1.24 g/g for p = 2.5 and 2.02 g/g for p = 1.0). The dehydrated myosin head is pear-shaped under the electron microscope, and its narrowest part is located near the junction with the tail [Elliott, A., & Offer, G. (1978) J. Mol. Biol. 123, 505-519]. Mendelson & Kretzschmar [Mendelson, R. A., & Kretzschmar, K.M. (1980) Biochemistry 19, 4103-4108] have shown that the pear-shaped molecule does not predict the experimental X-ray scattering curve. Nor is this model able to predict the hydrodynamic values. The three-dimensional model for S1 used by Mendelson and Kretzschmar gives a rather good fit to the experimental X-ray scattering curve, but it does not predict the hydrodynamic values. In order to try to reconcile the three models and to fit the X-ray scattering curve and the hydrodynamic data, we suggest that, in solution, the S1 monomer has the shape of a prolate ellipsoid and that an inclusion of bound water exists at one extremity of the protein. The rest of bound water surrounds the protein. As first approximation, the dry protein and the hole are assumed to have the same shape as the hydrated molecule (prolate ellipsoid; p).(ABSTRACT TRUNCATED AT 250 WORDS)     

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.     

Purification and properties of a plasminogen activator from cultured rat prostate adenocarcinoma cells

Zymographic analysis of the supernates from confluent cultures of a rat prostate adenocarcinoma cell line, PA-III, revealed the existence of two molecular forms of specific plasminogen activators, one of molecular weight of approximately 80 000 and another of approximate molecular weight of 45 000, in sodium dodecyl sulfate. The low molecular weight form has been purified 364-fold in 66% yield from the culture medium by a combination of gel filtration on Sephacryl S-200 and affinity chromatography on Sepharose 4B-benzamidine. The purified material possessed a specific activity of 192 000 urokinase CTA units mg-1. This enzyme displayed activity toward human Glu1-plasminogen, characterized by a Km of 1.7 +/- 0.2 microM and a Vmax of 0.53 +/- 0.1 pmol of plasmin min-1 unit-1. A synthetic chromogenic substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S-2288), was found for the activator. The enzyme possessed a Km of 0.33 mM and a kcat of 55 s-1 for S-2288. The activator was found to be a serine protease, inhibited by diisopropyl fluorophosphate (iPr2PF). At a concentration of 1 mM iPr2PF, and 30 nM enzyme, the half-time of this inhibition was 3.8 min. The 45 000 molecular weight enzyme was found to be inhibited by rabbit antibodies to human urokinase, thus characterizing the activator as a member of the urokinase class. The 80 000 molecular weight enzyme was not neutralized by anti-human urokinase but was neutralized by rabbit anti-human melanoma activator, likely allowing it to be classified as the tissue activator type.