Identical Electrophoretic Mobility Research Articles

Identification of two distinct amplifications of the esterase B locus in Culex pipiens (L.) mosquitoes from Mediterranean countries.

Two new highly active esterases were detected by starch electrophoretic studies in Culex pipiens mosquitoes from the area of Montpellier (France) and from Cyprus. We demonstrate here that both the French and the Cyprus esterases B are overproduced due to amplification of the coding gene. The production of the esterase B is approximately 50- and 500-fold higher in mosquitoes from France and Cyprus, respectively, than in susceptible insects, whereas the number of gene copies is about 25 and 250. Differences of about 7- and 95-fold were also found in the degree of chlorpyrifos resistance. RFLP comparison of the amplified region containing the esterase B gene revealed large differences between French and Cyprus mosquitoes. It thus appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified. We therefore named the haplotypes in mosquitoes from France and Cyprus B4 and B5, respectively. The estimated genetic distance between these two haplotypes is not smaller than those observed in all pair comparisons of other known esterase B haplotypes. These results are discussed in the context of amplification phenomena.

Ultrastructural Cytochemistry of Eosinophilic Inclusions in the Cells of Hyperplastic Nodules and Hepatomas in Mouse Liver

Intracisternal inclusions in the cells of 48 hyperplastic nodules and hepatomas in mouse liver were examined by electron microscopy to determine the precise compositions of the inclusions in relation to their ultrastructure, and some preliminary attempts at isolation and chemical analysis of the inclusions were performed. We classified the inclusions into two types. One was mainly of larger size, consisting of a single electron-lucent core and a granular cortical zone of high electron density. The other type was smaller, with a number of tiny, electron-lucent areas crowded into the central area instead of a single core. The cortical material of the inclusions was digested by pepsin treatment of thin sections, whereas the core and the electron-lucent areas within the cortical zone were not extracted. On the other hand, in materials treated with ethanol before post-osmication, only the core and electron-lucent areas within the cortical zone were partially extracted. The ultrastructure of the isolated inclusions was very similar to that of inclusions in situ. The chemical composition of the isolated fractions was estimated to be 60% protein and 35% lipid. Electrophoretically, the protein of this fraction showed a single band. We conclude that the cortical substance is proteinaceous in nature, probably consisting of a single protein or a group of proteins with identical electrophoretic mobility, whereas the core is composed of lipid. The possibility that the inclusions are due to an impairment in the mechanism of intracellular lipoprotein transport is discussed.

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation.

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.

Human papillomavirus type 18 E6*, E6, and E7 protein synthesis in cell-free translation systems and comparison of E6 and E7 in vitro translation products to proteins immunoprecipitated from human epithelial cells.

Expression of the E6 and E7 transforming genes of human papillomavirus type 18 (HPV18) occurs via structurally bicistronic mRNAs in which the downstream open reading frame (ORF) E7 is preceded either by the full-length ORF E6 or by a spliced ORF, E6*. We have used in vitro transcription and translation of HPV18 cDNAs in order to analyze the synthesis of E6*, E6, and E7 proteins and to compare the E6 and E7 in vitro translation products with the authentic proteins immunoprecipitated from cervical cancer cells. In wheat germ extract, in vitro translation resulted in the production of all three proteins, E6*, E6, and E7. In rabbit reticulocyte lysate, however, only the E6 and E7 proteins were produced. The lack of E6* protein was due neither to template RNA degradation nor to an inhibitory influence of the RNA 5' leader sequences, thus indicating the possibility of either inhibition of synthesis or degradation of E6* protein in reticulocyte lysate. The E7 protein was synthesized from both E6*-E7 and E6-E7 RNAs. In vitro-synthesized and authentic HPV18 E7 proteins revealed identical electrophoretic mobilities in two-dimensional gel electrophoresis, thus indicating similar modifications. By using a monoclonal antibody against the N terminus of HPV18 E6* and E6, an 18-kDa protein was detected not only in HPV18-positive but also in HPV18-negative epithelial cells. The 18-kDa proteins and the in vitro-synthesized HPV18 E6 protein exhibited comparable electrophoretic characteristics in two-dimensional gels. These results suggest the possible existence of a cellular protein related to HPV18 E6.

The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye

N,N-dipentadecylaminostyrylpyridinium iodide is a dye that is approximately 100-fold more intensely fluorescent in a lipid than aqueous environment. This observation suggests its potential as a fluorescence stain for lipoproteins. This work reports the staining of LDL with this dye for use in studies of cellular binding. The staining procedure is simple, resulting in stable attachment of the dye as determined by transfer experiments, physical properties essentially identical to native LDL as demonstrated by virtually identical electrophoretic mobility, and consistent results in studies of cellular binding using flow cytometry. Increased signal to noise ratio over other dyes used for lipoprotein staining including the widely used Dil (3,3′-dioctadecylindocarbocyanine iodide) allows determinations of greater sensitivity and precision to be made. This is demonstrated by the flow cytometric determination of the 4°C binding curve of LDL with freshly isolated human peripheral blood lymphocytes (i.e., cells not LDL receptor upregulated). Mediation of binding by the LDL receptor is demonstrated by correspondence between the LDL receptor dissociation constant derived from this work and literature values; increased specific binding in lymphocytes cultured in lipoprotein-deficient media to up-regulate the LDL receptor; and decreased specific binding in lymphocytes cultured in the presence of 25-hydroxy cholesterol for 48 h to suppress the LDL receptor.

Calcium-Dependent Phosphorylation of Symbiosome Membrane Proteins from Nitrogen-Fixing Soybean Nodules

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation.

Treatment of Data for Duplicated Gene Loci in Mixed-Stock Fishery Analysis

Isoloci (pairs of duplicated loci with a predominance of shared alleles of identical electrophoretic mobility) are common in salmonid fishes and present special problems for the analysis of mixed-stock fisheries. The method currently used to treat isolocus data (which assumes that allele frequencies at both loci are the same) leads to bias if the true frequencies (P, Q) are not identical. Using simulated mixtures drawn from populations with (1) hypothetical allele frequencies and (2) allele frequencies observed at four isolocus systems in samples of chinook salmon from the Fraser River system, we show that a better method is to use the maximum likelihood estimates ([Formula: see text], [Formula: see text]) of allele frequencies at the individual loci. In simulations in which the population frequencies at the two loci of an isolocus pair differed, use of ([Formula: see text], [Formula: see text]) resulted in less error in estimating the true mixture proportions than did use of a single average frequency, whereas the two methods produced very similar results if population frequencies were identical at both loci.

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

Effect of polymer ion concentrations on migration velocities in ion-exchange electrokinetic chromatography

Ion-exchange electrokinetic chromatography was developed for the separation of analyte ions having identical electrophoretic mobilities in capillary electrophoresis. The separation principle is based on the differential ion-pair formation of the analyte ion with a polymer ion. Polybrene and poly(diallyldimethylammonium chloride) were employed as polymer ions and some isomeric acids as analytes. The dependence of the relative migration velocities of the analytes on the concentration of the polymer ions was studied experimentally, and the results are discussed in comparison with a theoretically derived equation.

Glutaraldehyde crosslinking analysis of the C 12E 8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C 12E 8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K +-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K +-stimulated hydrolysis with a K 0.5 of 28 ± 4 μM ATP, and inhibition with an IC 50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K + was also observed with a K 0.5 for activation of 2.8 ± 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC 50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K +-stimulated activity with a K i of 0.035 μM. The soluble enzyme was stable with a t 0.5 for ATPase activity of 6 h between 4 and 11°C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w / w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10–35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.

Comparative action of calpain on erythrocyte Ca2+-pumping ATPase in sickle cell anaemia, essential hypertension and kwashiorkor

Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca2+, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca2(+)-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca2(+)-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca2(+)-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.

Secretion of methanol-insoluble heat-stable enterotoxin (STB): energy- and secA-dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin

The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extracellular polypeptide. The toxin (STB) was first detected as an 8,100-Mr precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form. Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background. After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent. The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant. The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence. The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.

Electrophoretic Characterization of Quinoa Seed Proteins

AbstractQuinoa (Chenopodium quinoa Willd) is an important domesticated food crop of the Andean highlands with potential as an alternative crop elsewhere. Among its most attractive characteristics are the quantity and favorable amino acid balance of the seed proteins. The objective of this study was to characterize quinoa seed proteins by electrophoretic mobility, solubility fractionation, and genetic variability from a wide genetic base. Electrophoretic profiles of denatured albumin, globulin, prolamin, and glutelin solubility fractions demonstrated that quinoa seed polypeptides could be classified as either albumin or globulin with most predominant polypeptides in the globulin fraction. Insignificant amounts of protein were present in the prolamin fraction and all polypeptides in the glutelin fraction had identical electrophoretic mobilities to albumins and globulins. Three globulin polypeptides of 34.3, 35.6, and 36.2 kilodaltons in size were highly variable within and among the accessions examined and appear to be coded by at least two loci. Two‐dimensional peptide mapping revealed that these three polypeptides were homologous. These highly variable markers could be used for identification and classification of germplasm and elucidation of systematics and genetic variability within the quinoa germplasm pool. All other major polypeptides were electrophoretically invariant among the accessions examined.

Comparisons of the Genomic RNA of Arkansas DPI Embryonic Passages 10 and 100, Australian T, and Massachusetts 41 Strains of Infectious Bronchitis Virus

The genomic ribonucleic acid (RNA) of Arkansas DPI at embryonic passages 10 and 100, Australian T, and Massachusetts 41 strains of infectious bronchitis virus (IBV) were compared using two-dimensional, 32PpCp-labeled oligonucleotide fingerprinting. The Arkansas DPI strain embryonic passage 10 was pathogenic for chickens and had one oligonucleotide not present in the attenuated passage 100. The remaining Arkansas DPI oligonucleotides had identical electrophoretic mobility. The fingerprint patterns of the Australian T, Massachusetts 41, and Arkansas DPI IBV genomes were dissimilar, thus indicating they were genetically distinct. The distinctive fingerprint patterns of these virus strains, all identified as IBV by other criteria, indicate considerable genomic variation. Fingerprinting is reliable and sensitive, and should be used in combination with other characterization methods for identifying and differentiating IBV strains.

Ion-exchange electrokinetic chromatography with polymer ions for the separation of isomeric ions having identical electrophoretic mobilities

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIon-exchange electrokinetic chromatography with polymer ions for the separation of isomeric ions having identical electrophoretic mobilitiesShigeru. Terabe and Tsuguhide. IsemuraCite this: Anal. Chem. 1990, 62, 6, 650–652Publication Date (Print):March 15, 1990Publication History Published online1 May 2002Published inissue 15 March 1990https://doi.org/10.1021/ac00205a022RIGHTS & PERMISSIONSArticle Views161Altmetric-Citations131LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (386 KB) Get e-Alerts Get e-Alerts

Isolation and characterization of six pathogenesis-related (PR) proteins of Samsun NN tobacco

The purification to homogeneity of pathogenesis-related (PR) proteins R and S from Nicotiana tabacum cv. Samsun NN leaves has been achieved by using a combination of conventional and high-performance chromatographic supports. The same procedure allowed the purification and the characterization of four other proteins which displayed some properties characteristic of tobacco PR proteins and were shown to accumulate in tobacco leaves in response to virus infection. They can be, therefore, considered as new tobacco PR proteins which we designate as PR-s1, -s2, -r1 and -r2. The relative electrophoretic mobilities (Rf) under non-denaturing conditions were estimated to 0.30 for PR-r1 and -r2, 0.25 for Pr-R, 0.20 for PR-s1 and -s2 and 0.15 for PR-S. On SDS gels PR proteins R and S possessed the same apparent molecular weight (Mr 24,000) as did PR-proteins s1 and r1 (Mr 14,500) and PR-s2 and -r2 (Mr 13,000). However, proteins s1, s2, r1 and r2 had identical electrophoretic mobilities on SDS gels when the loading sample buffer contained no reducing agent. Polyclonal antisera were raised against PR proteins R and S and used in immunoblotting experiments. Proteins R and S were shown to be serologically closely related. No cross-reaction was detected with any of the four new tobacco PR proteins r1, r2, s1 and s2 or with the previously described PR proteins, i.e. PR-1a, -1b, -1c, -2, -N, -O, -P and -Q.

A high molecular weight subunit of wheat glutenin seed storage protein correlates with its flour quality.

The high molecular weight (HMW) subunits of wheat seed storage proteins in 376 cultivars were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The highest molecular weight subunit designated as 145 kD subunit was found frequently in Japanese cultivars (Fig. 1). This subunit has identical electrophoretic mobility of the glutenin high molecular weight subunit 2.2 as reported by PAYNE et al. (1983). The 145 kD subunit separated by SDS-PAGE was electroblotted onto a polyvinylidene difluoride membrane filter and the blotted subunit was sequenced by a gas-phase sequencer. The N-terminal amino acid sequence of the 145 kD subunit determined was consistent with that of the HMW glutenin subunits reported previously (Table 1). The 145 kD subunit was concluded to be one of the glutenin seed storage proteins. The size and number of hard crystal particles which characterize hard wheat flours (Fig. 2), varied depending on the presence or absence of the glutenin 145 kD subunit (Table 2); The cultivars without the 145 kD subunit always produce hard flours. The frequency of cultivars with the 145 kD subunit was higher in the southern part of Japan than the northern part (Fig. 3); Most of the cultivars predominant to the northern part of Japan (Hokkaido, Tohoku, Hokuriku and Nagano Districts) have no 145 kD subunit but many large crystal particles in the flours. However, most of the cul-tivars in the south Japan (Kyushu District) have this subunit but not many large crystal particles. These results suggest that the 145 kD subunit plays an important role in determining the hardness of wheat flours. The crossing test revealed that the expression of the 145 kD subunit is controlled by a single gene (Table 3, Fig. 4). The trace back of pedigree shows that the genotypes with and without the 145 kD subunit were preferably selected in each step of the wheat breeding procedures in the southern and northern part of Japan, respectively (Fig. 5).

N-acetylation pharmacogenetics: a gene deletion causes absence of arylamine N-acetyltransferase in liver of slow acetylator rabbits.

The New Zealand White rabbit provides a widely used animal model for the human acetylation polymorphism, which confers marked interindividual variation in the effect and toxicity of numerous drugs, chemicals, and potential carcinogens. The relationship of a recently isolated cDNA clone, designated rnat, to genetically polymorphic arylamine N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) of rabbit liver was established by its expression in monkey kidney COS-1 cells: (i) cytosols from transfected cultures contained high levels of an Ac-CoA-dependent NAT activity, which was kinetically indistinguishable from that observed in cytosols from livers of genetically rapid-acetylator rabbits; (ii) transfected cells also contained an immunoreactive protein, recognized by NAT-specific antibodies, with identical electrophoretic mobility to NAT from rabbit liver. The rnat clone and anti-NAT antibodies were then used to study the relationship between NAT activity, liver enzyme protein, and the level of mRNA in livers from in vivo phenotyped rapid- and slow-acetylator rabbits. Livers from slow acetylators were devoid of both immunodetectable NAT protein and its corresponding mRNA. Analysis of genomic DNA with a panel of restriction enzymes revealed the loss of specific hybridizing bands in the DNA of slow-acetylator rabbits. These data strongly suggest that defective arylamine N-acetylation in the rabbit model is caused by a gene deletion resulting in an absence of specific mRNA and NAT enzyme protein.

Characterization of human neutrophil glycoproteins expressing the CD15 differentiation antigen (3‐fucosyl‐N‐acetyllactosamine)

The expression of the CD15 antigen, 3-fucosyl N-acetyllactosamine, on neutrophil glycoproteins has been studied by SDS gel electrophoresis and immunoblotting. The antigen is expressed on several glycoproteins, both intracellularly and on the cell surface. Each subcellular compartment appears to contain a specific antigen. A soluble, granule glycoprotein (Mr 80-90K) probably accounts for most of the intracellular staining detected immunohistochemically. Membrane glycoproteins of Mr, 85-90K and 25K are associated with granule membranes, the latter being an integral membrane protein. The CD15 antigen is expressed on several cell surface glycoproteins with Mr in the range of 165K and 105K. These antigens are also contained in an intracellular pool which is brought to the surface on activation of the cells with chemotactic peptides. The 165K and 105K antigens show identical electrophoretic mobility to two of the major glycoproteins detectable by PAS or protein staining of gels of detergent extracts of cell membranes. These glycoproteins include the complement receptor, CR3. The beta chain of CR3 (105K) and to a lesser extent the alpha chain (165K) express CD15; however, most of the CD15 antigen is associated with other glycoproteins of these molecular masses.

Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues

Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue.