Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB ⁻ cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.     

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

Metabolic oxygen regulation and conformity during submergence in the salamandersSiren lacertina,Amphiuma means,andAmphiuma tridactylum,and a comparison with other giant salamanders

Summary The giant salamanders of North America include 4 genera, all of which are aquatic. We have compared the efficacy of aquatic O₂ uptake among them by measuring theVO₂ while submerged and determining the responses to progressive hypoxia at 10–240 mmHg at 20° C. Both species ofAmphiuma were metabolic O₂ conformers over the entire range ofPO₂. About half ofSiren lacertina were conformers over this range, and half were regulators with an average critical O₂ tension of 92 mmHg. There were no short-term changes (days) in the response ofSiren to progressive hypoxia, but one animal switched from conformation to regulation after 4–5 months. Neither genus is considered to have an exceptionally low metabolic rate. The “whole-body O₂ conductance”, defined asΔVO₂/ΔPO₂(μl O₂ · g⁻¹ · h⁻¹ · mmHg⁻¹) in the range of metabolic O₂ conformity, was least in the species most dependent upon air-breathing and most likely to be found in hypoxic waters (e.g., 0.076 forAmphiuma), and greatest in those that airbreathe less frequently and/or are found in relatively normoxic waters (e.g., 0.429 forNecturus). These conductances are considered to be adaptive in terms of preventing O₂ loss through the skin, or in facilitating its uptake, as correlated with the O₂ tensions normally prevailing in the environment of each species.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

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.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

Probabilistic reconstruction of ancestral protein sequences

Using a maximum-likelihood formalism, we have developed a method with which to reconstruct the sequences of ancestral proteins. Our approach allows the calculation of not only the most probable ancestral sequence but also of the probability of any amino acid at any given node in the evolutionary tree. Because we consider evolution on the amino acid level, we are better able to include effects of evolutionary pressure and take advantage of structural information about the protein through the use of mutation matrices that depend on secondary structure and surface accessibility. The computational complexity of this method scales linearly with the number of homologous proteins used to reconstruct the ancestral sequence.     

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.