Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). I. Comparative electrophoretic studies on blood enzymes and proteins in Arctic and silver foxes

Twenty-seven blood enzymes and proteins, whose structures are presumably controlled by at least 33 genes, were assayed in Arctic and silver foxes by starch gel electrophoresis. Two types of protein and enzyme electrophoretic patterns were distinguished: one exhibiting a single enzyme, the other several isozymes. The two fox species were found to differ in seven of the 27 enzymes and proteins studied: glucose-6-phosphate dehydrogenase, adenylate kinase, erythrocyte carboxylesterase, diaphorase, prealbumin, transferrin, and albumin. No differences were established between the species for the other enzymes and proteins. The data are interpreted as evidence for the existance of a set of enzymes and proteins differentiating the Arctic from the silver fox.     

An estimation of the degree of the genetic divergence of sibling species Microtus arvalis and Microtus subarvalis (Rodentia) based on electrophoretic analysis

The electrophoretic mobilities of 52 enzymes and proteins were used as measures of the genetic similarity between the sibling species Microtus arvalis and M. subarvalis. The two vole species differed in the electrophoretic mobilities of seven (glucose-6-phosphate dehydrogenase, adenylate kinase, diaphorase, lactate dehydrogenase-A, alpha-galactosidase, 6-phosphogluconate dehydrogenase, and hemoglobin) of these markers. This allowed us to accept the seven markers assayed as species-specific markers. Based on the frequency distribution of the genes at the polymorphic loci of M. arvalis and M. subarvalis, the degree of their genetic similarity was estimated as 0.312 and the genetic distance as 1.164 by Nei's formula. The estimates for genetic similarity were close to those obtained for species recognized as distinct.     

Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects

Carboxylesterases provide key mechanisms of resistance to insecticides, particularly organophosphates (OPs), in insects. One resistance mechanism is a qualitative change in the properties of a carboxylesterase. Two mutant forms, G151D and W271L, have been observed, mostly in dipteran species, to affect substrate specificity of enzymes. But whether these two single mutations can commonly change character of insect carboxylesterases is unknown. In our study carboxylesterase genes from seven insects distributed among four orders were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. The kinetics of the purified recombinant proteins was examined towards an artificial carboxylester and two OP insecticides. The G/A151D and W271L mutation significantly reduced carboxylesterase activity in 87.5% and 100% cases, respectively, and at the same time conferred OP hydrolase activities in 62.5% and 87.5% cases, respectively. Thus, the change at position 271 is more effective to influence substrate specificity than that at position 151. These results may suggest that these two mutations have the potential to cause insecticide resistance broadly in insects.     

Genetics of octanol and α-glycerophosphate dehydrogenases in the mosquito Aedes (Finlaya) togoi

Genetic studies were performed on octanol dehydrogenase (EC 1.1.1.73) and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8) in the mosquito Aedes (Finlaya) togoi by agar gel electrophoresis. The electrophoretic survey revealed two octanol dehydrogenase loci (Odh-1, Odh-2) and one alpha-glycerophosphate dehydrogenase locus (alpha-Gpdh) in this species. Five alleles were observed at the Odh-2 locus in seven laboratory strains, whereas the alpha-Gpdh locus was completely monomorphic in six of seven strains examined and the second allele at this locus was detected in only one strain at a frequency of 0.14. Both loci code for dimeric enzymes. Linkage studies on Odh-2 and alpha-Gpdh suggested that the gene arrangement and recombination units were Odh-2--(25.8)--M (sex locus)--(30.5)--s (strawcolored larva) and M--(25.6)--alpha-Gpdh--(15.4)--s. These results, together with linkage data previously reported, give the following gene linkage on the sex chromosome: Odh-2--Est-3 (carboxylesterase)--Acph (acid phosphatase)--M--alpha-Gpdh--s--Est-2 (carboxylesterase). The total map length of this arrangement is approximately 75 map units.     

Diaphorase P: a new fetal isozyme identified in human placenta

Human placenta contains a thermostable, cytosolic NADH-diaphorase which is different from the other diaphorases and which we designate as diaphorase P. It is specific for NADH and reduces artificial substrates such as dichlorophenol and tetrazolium derivatives, but not natural substrates such as methemoglobin, cytochrome b5 or lipoate. It is antigenically distinct from the ubiquitous red-cell type NADH-diaphorase (soluble cytochrome b5 reductase) specified by the DIA1 locus. Using electrophoretic and immunologic methods, it was possible to detect diaphorase P in various fetal tissues (brain, liver, kidney, muscle), whereas was not found in adult tissues with the exception of the brain. This enzyme, the physiological role of which remains unknown, appears to belong, therefore, to the category of fetal proteins. Its resurgance in primary liver cancer was demonstrated in three cases.     

Purification and identification of cutinases from <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis> and <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.     

Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non-denaturing two-dimensional gel electrophoresis

After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.     

Prostatic acid phosphomonoesterase of the Japanese monkey (Macaca f. fuscata)

Possible immunological differences between monkey and human prostate gland proteins and also between seminal vesicle proteins of these species were investigated by agarose gel electrophoresis, agarose gel immunoelectrophoresis and by the agar gel immunodiffusion method, using anti-sera against human plasma, human seminal plasma and human prostatic acid phosphomonoesterase (PMEase). At the same time, the electrophoretic mobility of these prostatic acid PMEases was compared by means of starch gel electrophoresis.Each of these two tissues, monkey and human, was found to contain antigenic proteins with immunological identity. Though antigenic similarity of monkey and human prostatic acid PMEase was demonstrated by immunological methods, a clear difference was observed in the electrophoretic mobility of these enzymes when examined by starch gel electrophoresis.     

Distances électrophorétiques entre les cinq espèces de babouins du genre Papio basées sur les mobilités des protéines et enzymes sériques

The baboons of the Papio genus have for a long time been divided into five distinct species, P. papio, P. anubis, P. cynocephalus, P. ursinus and P. hamadryas, with the latter being classed apart from the other four. This classification has been based on morphological, sociological and biogeographical grounds. Recently, however, the five forms have been considered as merely different sub-species of a polytypic species. This view has been supported by calculations of genetic distances between four of the species, the data being based on the electrophoretic polymorphism of the erythrocytic enzymes. The overall distances calculated are not greater than those obtained for the subspecies of other vertebrate genera. The present article shows that P. papio and P. anubis are indistinguishable in this respect, but that differences between the four species are observed by measurements of the electrophoretic mobilities of at least seven proteins and serum enzymes, namely: albumin, transferrine, esterases C₁ to C₄ and alkaline phosphatase. The apparent contradiction between these results may be explained in two ways. Firstly, the genetic distances correspond to a relatively low level of individual heterozygosity. The second, and principal reason, is that the proteins and serum enzymes present in the various tissues have evolved much more rapidly with time than the intracellular erythrocytic enzymes. Seven proteins can therefore be used to distinguish between the four species. The differential electrophoretic mobility data have been treated by the classical methods of multidimensional analysis to give a planar projection of the distances between the species. This shows P. hamadryas to be the most distinct form. The results obtaind agree with those derived from studies of the immunology of the transferris, the isoantigens, and from DNA hybridization experiments.     

Histochemical investigation of some mitochondrial and microsomal enzymes in the kidneys of rabbits immunized with type B botulinus anatoxin.

Immunization of rabbits with botulinus anatoxin containing a number of proteins of bacterial origin causes a statistically significant increase in the activity of succinate dehydrogenase, NAD diaphorase and NADP diaphorase as early as after 24 hours. After 5-7 days, the activity of all mitochondrial enzymes drops below the control level and returns to normal by the 14th day. The activity of glucose 6-phosphatase decreases significantly already 24 hours after immunization and returns to normal by the end of the 7th day. The mechanism of excretion of foreign protein in the kidneys of immunized animals is discussed.     

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.     

Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome

Liver microsomes are subcellular fractions that contain many metabolizing enzymes for drugs and endogeneous compounds. Some of these enzymes are regulated by sex hormonal control and exhibit sex-dependent expression pattern and metabolizing speed. Studying these enzymes, however, are complicated by the presence of isoforms such as cytochrome P450 (CYP450), which families share more than 50% amino acid identities. In this study, we applied quantitative shot-gun proteomics approach coupled with stable-isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. Although shot-gun approach is rarely used for identifying protein isoforms, we identified 53 isoforms by at least one unique peptide including 21 isoforms of CYP450s. Moreover, by quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes including CYP2C12 CYP2C11, CYP2C13, CYP2B3, CYP2C11, CYP2C70 and CYP3A2 which are known to be male specific, 21 female dominant enzymes including CYP2A1, CYP2C7, CYP2C12, CYP2D26, alcohol dehydrogenase 1, carboxylesterase 3, glutathione S-transferase, liver carboxylesterase 4, UDP-glucuronosyltransferase 2B1, and glyceraldehyde-3-phosphate dehydrogenase which are known to be female specific; and 125 sex-independent enzymes. However, most of the sex specificities revealed from this study, such as the male specificity of CYP2D1, were novel and not yet reported. We then conducted a mass spectrometry-multiple reaction mode (MS-MRM) based enzyme activity method to determine the catalyzing rate of CYP2D1 in male and female liver microsomes using carteolol as its specific substrate. The reaction rate catalyzed by CYP2D1 in female rats was determined to differ significantly with the rate in male rats. Moreover, the ratio (female/male) of reaction rate (0.68) was found to correlate with their relative protein abundance (0.72). This study revealed novel sex dependences of many rat liver enzymes and also demonstrated a unique MS-based analytical platform that could identify novel iso-enzymes and further quantify their abundance and enzyme activity.     

Cloning and sequencing of a human liver carboxylesterase isoenzyme

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.     

Comparative Analysis of Rapidly Transported Axonal Proteins in Sensory Neurons of the Frog and Rat

³⁵S-labeled proteins carried by fast axonal transport in sciatic sensory axons of bullfrog and rat were separated electrophoretically on discontinuous polyacrylamide gradient slab gels. In contrast to the previously reported similarity in the electrophoretic profiles of rapidly transported proteins from functionally different neurons, we have found that there is very little correspondence in the profiles of these proteins in functionally similar neurons from two widely studied species. We also found very little correspondence between the two species in the profiles of locally synthesized sciatic nerve protein. The results demonstrate the difficulty inherent in comparing the electrophoretic profiles obtained using these two model systems for the study of rapidly transported axonal proteins. In particular, relationships between the major rapidly transported proteins in the two species could_not be analyzed with this technique.     

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues

 The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes – neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms. Accepted: 2 September 1996     

The amino acid substitution and some chemical properties of a variant human erythrocyte carbonic anhydrase: Carbonic anhydrase IdMichigan

Carbonic anhydrase Id_Michigan, an electrophoretic variant of human red cell carbonic anhydrase I, was purified from erythrocytes obtained from an individual heterozygous for the trait. Primary structural analysis indicates that a lysine residue has exchanged for a threonine residue in the variant enzyme. After isolation, there was approximately 1.8 times as much normal as variant enzyme. Thermostability studies demonstrated that carbonic anhydrase Id was more thermolabile than the normal enzyme. The normal and variant enzymes showed no differences in specific carboxylesterase activity or CO₂ hydratase activity. Utilizing the carboxylesterase activity toward -naphthyl acetate, the normal and variant enzymes had similar Michaelis constants, pH profiles, and rates of inhibition by acetazolamide. Immunochemical studies did not demonstrate an antigenic difference for the variant enzyme.Supported in part by Research Grants 2 T1 GM-76, 5 TO1 GM 00071-09, and GM 09252 from U.S. Public Health Service.This report is a portion of a dissertation submitted to the University of Michigan in partial fulfillment of the requirements for the doctor of philosophy degree.     

Electrophoretic and morphological comparisons in ten rain forest species of Shorea (Dipterocarpaceae)

Differences identified by electrophoretic separation of enzymes and other proteins are providing increasing informative for the study of evolutionary affinities within a genus. Zymogran compared with a scheme of putative affinities derived from a standard taxonomic classi based on consciously weighted morphological characters, as well as with indices of similarity on an array of morphological criteria relating to twigs, stipules, leaves, inflorescences and structure for 10 species of Shorea from southeast Malaysia. Shorea ovalis is placed in a separate of the genus from the other nine species; S. leprosula, S. parviflia and S. curtisii are related other on R^p data and a high degree of similarity is shown between these three spec S. dasyphylla .     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

Comparison of cell wall and membrane proteins from eight Candida species

Cell surface proteins from eight Candida species were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell wall proteins of these species were similar in their electrophoretic patterns. On the other hand, differences were detected among the patterns of membrane proteins, although C. albicans serotypes A and B gave similar electrophoretic patterns, and so did two species (C. pseudotropicalis and C. parapsilosis).     

Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids

The production of four liver-specific enzymes (tyrosine aminotransferase or TAT, alanine aminotransferase, aldolase B, and alcohol dehydrogenase) has been analyzed in rat hepatoma-mouse lymphoid cell hybrids containing a 1s or 2s complement of rat chromosomes. The hybrid clones which retain a nearly 2s complement of rat chromosomes show high activity of all four enzymes; those which contain a 1s rat complement show partial or complete extinction of these enzymes. The tyrosine aminotransferase produced by most of the hybrid clones is identifiable as being of both rat and mouse origin, based upon criteria of temperature sensitivity and electrophoretic mobility; antiserum to the rat liver enzyme was used to distinguish the pseudo-TAT produced by the lymphoid parent from liver-TAT produced by hepatoma and hybrid cells. By the criteron of electrophoretic mobility, the liver form (B) of aldolase, produced by only some of the hybrid clones, appears to be composed of both rat and mouse subunits. We conclude that when extinction of tissue-specific proteins does not occur or is only partial in hybrid cells (due to gene dosage effects), the genomes of both parents may be active in directing synthesis of these proteins.