Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Sequence and expression of a human type II mesothelial keratin

Using mRNA from cultured human mesothelial cells, we constructed bacterial plasmids and lambda phage vectors that contained cDNA sequences specific for the keratins expressed in these cells. A cloned cDNA encoding keratin K7 (55 kD) was identified by positive hybrid selection. Southern Blot analysis indicated that this sequence is represented only once in the human genome, and Northern Blot analysis demonstrated that the gene encoding K7 is expressed in abundance in cultured bronchial and mesothelial cells, but only weakly in cultured epidermal cells and not at all in liver, colon, or exocervical tissue. The predicted amino acid sequence of this keratin has revealed a striking difference between this keratin and the type II keratins expressed in epidermal cells: whereas all of the epidermal type II keratins thus far sequenced have long nonhelical termini rich in glycine and serine, this mesothelial type II keratin has amino and carboxy terminal regions that are unusually short and lack the inexact repeats of glycine and serine residues.     

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).     

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.     

Quantitative field investigations of feeding and territorial behaviour of young-of-the-year brook charr,Salvelinus fontinalis

Synopsis Direct observations of young-of-the-year brook charr, Salvelinus fontinalis, in a second-order woodland stream indicated that most of their feeding effort was directed toward sub-surface, drifting prey (83% of feeding time). Feeding from the substrate and water surface was much less frequent (17% of feeding time). Comparisons of gut contents to drift net and substrate fauna samples corroborated that the most commonly consumed prey (chironomid and trichopteran larvae, ostracods, and ephemeropteran nymphs) were captured primarily from sub-surface, invertebrate drift. The disproportionate numbers of some prey species in the guts of several fish indicate that some prey selection occurred. Territories appeared to be cardioid-shaped, and were often contiguous, with dominance hierarchies evident among the residents. Agonistic interactions were frequent. Charges and chases predominated (91% of interactions) while lateral displays were infrequent (9% of interactions). Overall, these fish spent most of the daylight hours station-holding (77%) and feeding (18%). While only 3% of total time was spent in aggression, this amounted to 14% of the time a fish spent away from its station. There was some indication that territories were defended at a cost of feeding time.     

Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.