Electrophoretic Forms Research Articles

Zein of maize grain: II — the charge heterogeneity of free subunits

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 4.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.

Isoelectric forms of clusterin isolated from ram rete testis fluid and from secretions of primary cultures of ram and rat Sertoli-cell-enriched preparations.

Clusterin, a cell aggregating factor isolated from ram rete testis fluid (RTF), is shown to contain 14.7% hexoses, 13.6% glucosamine, and 7.9% sialic acid. The isoelectric point (pI) of the predominant electrophoretic form of clusterin from ram RTF is 3.7. After treatment with neuraminidase, the pI values become more basic, with the majority of the material being eluted from a chromatofocusing column at pH values between 4.9 and 5.1. Intact clusterin binds quantitatively to wheat germ agglutinin - Sepharose 6 MB, but after treatment with neuraminidase only 49% specifically binds. Clusterin isolated from proteins secreted by primary cultures of ram Sertoli-cell-enriched preparations was shown to have properties similar to those of intact clusterin isolated from ram RTF. In contrast, clusterin isolated from proteins secreted by primary cultures of rat Sertoli- or granulosa-cell-enriched preparations has isoelectric forms which more closely resemble those of neuraminidase-treated ram clusterin.

Purification, properties and limited proteolysis of nitrate reductase from Pseudomonas denitrificans

The membrane-bound nitrate reductase (nitrate: (acceptor) oxidoreductase, EC 1.7.99.4) of Pseudomonas denitrificans ATCC 13867 solubilized either by treatment with sodium deoxycholate plus ammonium sulfate (nitrate reductase I) or by neutral heat treatment (nitrate reductase II) was purified to homogeneity. The molecular weights of nitrate reductase I ( s 20,w = 11.2 S) measured by gel filtration and sedimentation equilibrium methods were 220 000 and 230 000, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicates that nitrate reductase I is composed of three different subunits with molecular weights of 136 000, 55 000 and 19 000. Nitrate reductase I was found to contain (in relative molar amounts): 1.0 heme, 0.74 molybdenum, 13 non-heme iron, and 12 acid-labile sulfide. Neither FAD nor FMN was detected. The molecular weights of nitrate reductase II ( s 20,w = 9.6 S) measured by gel filtration and sedimentation equilibrium methods were 190 000 and 200 000, respectively. Nitrate reductase II was shown electrophoretically to be composed of two different subunits with molecular weights of 136 000 and 55 000. Nitrate reductase I was converted to nitrate reductase II by heat treatment in alkaline solution or by aging. The composition of nitrate reductase II was essentiatlly the same as that of nitrate reductase I, except that heme was not detected. Some enzymological properties are reported. The enzyme was shown to contain the molybdenum cofactor. Oxidation products of the molybdenum cofactor gave pterin-6-carboxylic acid and 6-carboxypterin-7-sulfonic acid upon alkaline KMnO 4 oxidation. During storage of 4°C for about a month, a purified preparation of nitrate reductase II was converted to a new electrophoretic form, the molecular weight of which was approx. 169 000. Electrophoretic analysis of freshly prepared and stored enzyme indicated conversion of the small subunit ( M r 55 000) to a 43 kDa polypeptide and partial cleavage of the large subunit ( M r 136 000) to 87 kDa and 47 kDa fragments. Similar polypeptide fragments were obtained by incubation of the enzyme with trypsin. The trypsin-treated enzyme ( M r 169 000) retained full enzymatic activity. Despite the tryptic cleavage, the three fragments appeared associated upon gel chromatography.

Effect of methylamine and plasmin on the conformation of human alpha 2-macroglobulin as revealed by differential scanning calorimetric analysis

Differential scanning calorimetric analysis was used as a probe of the conformational alteration in human alpha 2-macroglobulin (AM) upon its complex formation with methylamine and with the protease, human plasmin. The slow electrophoretic form of AM displayed a single thermal transition, characterized by a temperature midpoint (Tm) of 65.8 +/- 0.3 degrees, a calorimetric enthalpy (delta Hc) of 2,550 +/- 150 kcal/mol and a van't Hoff enthalpy (delta Hvh) of 140 kcal/mol. In the presence of sufficient methylamine to irreversibly disrupt the four thiol ester bonds in AM, a single thermal transition was obtained, characterized by a Tm of 62.8 +/- 0.3 degrees, a delta Hc of 1,700 +/- 100 kcal/mol, and a delta Hvh of 169 kcal/mol. These data suggest that a major conformational alteration is produced in AM upon complex formation with methylamine. When plasmin interacts with AM, the resulting thermogram displays Tm values for AM of 68-69 degrees and 77 degrees, also suggestive of a large conformational alteration in AM. However, this latter alteration appears dissimilar to the change induced by methylamine.

Calcium-activated ATPase of the chick embryonic chorioallantoic membrane. Identification, developmental expression, and topographic relationship with calcium-binding protein.

A Ca2+-activated ATPase activity is present in the chick embryonic chorioallantoic membrane (CAM), the placenta-like tissue which translocates eggshell calcium into the embryonic circulation. The enzyme is membrane-bound, ATP-specific, Mg2+-dependent, exhibits dual Km values of 30 microM and 0.3 mM Ca2+, and has a Mr of 170,000. Throughout embryonic development, a single electrophoretic form of the Ca2+-ATPase is found and, furthermore, its specific activity as a function of age follows a bimodal pattern. In particular, from incubation days 14-15 to the end of gestation, a period representing rapid embryonic calcium accumulation, Ca2+-ATPase specific activity increases 6-fold. Cytohistochemistry localized the Ca2+-ATPase exclusively within the CAM ectoderm which lies adjacent to the calcium-rich shell membrane/eggshell. In a parallel study, cleavable bifunctional cross-linking agents were used to characterize the in situ protein topography of the CAM ectodermal surface adjacent to the calcium-binding protein (CaBP), a CAM cell-surface protein associated with calcium transport. We found that the immediate near neighbor of the CaBP is a 170,000 Mr, membrane-bound protein. The 170,000 protein was co-isolated with the CaBP after cross-linkage in situ and subsequent immunoprecipitation with anti-CaBP antibodies. Reductive cleavage of the immune complex released detectable Ca2+-ATPase activity, suggesting that the 170,000 protein is the Ca2+-ATPase of the CAM.

Amylase secretion by sweet potato, Ipomoea batatas (L.) Lam., cell cultures grown on various carbon sources

Cell cultures of sweet potato grown in media containing sucrose, glucose, maltose, or starch secreted amylase into the growth medium. The growth rate of cells was not appreciably affected by the carbon source employed for growth, although cells grown on sucrose had a slightly longer lag period before exponential growth occurred. Amylase levels inside the cells were not affected by carbon source, but the amount of amylase released into the medium was drastically affected. Maltose-grown cells released the most amylase while sucrose-grown cells released the least. Cells grown in the light released about twice as much amylase as cells grown in the dark when grown on glucose, maltose, or starch. Three amylase electrophoretic forms were found in the storage root tissue from which all cultures were derived. Cells grown in culture exhibited either two or three amylase forms, depending on the carbon source. The slowest migrating root amylase was found only in cells grown on starch. The root amylase having intermediate mobility was present in all cultures, as was a form having higher mobility than the most mobile root form. The fastest migrating electrophoretic form from the root was not present in any of the cells.

Tetrathionate-blocked creatine kinase as a substrate for human plasma ‘creatine kinase conversion factor’

‘Creatine kinase conversion factor’ has been partially purified from whole plasma. The preparation has the ability to convert slowly migrating, native CK-MM into a faster electrophoretic form through a form of intermediate mobility. These changes are accompanied by a loss in CK activity. The rates of both electrophoretic conversion and loss of enzyme activity are reduced or prevented by the presence of 2-mercaptoethanol, EDTA, or by formation of the creatine-MgADP-nitrate dead-end complex. When the thiol group essential for enzyme activity found at the CK active site is first blocked by treatment with potassium tetrathionate, full activity may be retained under conditions that fully convert CK to the faster electrophoretic forms. The conversion factor activity was not inhibited by a pre-incubation with the protease inhibitor, PMSF and the converted form of CK showed no evidence of proteolytic cleavage. The molecular basis of the chemical alteration caused by the conversion is discussed.

Changes in the electrophoretic pattern of salivary peroxidase at the middle of the menstrual cycle

Salivary peroxidase activity is known to increase at the middle of the menstrual cycle, and we report changes in electrophoretic patterns of salivary peroxidase over the same period. Peroxidase during the early follicular and late luteal phases of the menstrual cycle has been resolved into three forms by polyacrylamide gel electrophoresis. An additional, low mobility peroxidase has been detected at midcycle when two electrophoretic forms occurring at other times are reduced; this is coincident with the peak in total perosidase activity. Available evidence suggests that this ovulatory peroxidase represents a catalytically active aggregate of the peroxidases normally present in saliva.

MRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase.

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.

Oocyte specific regulation of PGK-1 isozyme activity in female germ cells of the mouse

SUMMARYThe expression of electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1) was examined during the ontogeny of the female germ-line followingX-chromosome reactivation. Non-growing oocytes from foetal and neonatal ovaries of heterozygous females show a higher PGK-1A isozyme activity, a reflection of prior non-randomX-inactivation favouring activity of theX-chromosome carrying thePgk-1aallele andXceclocus. On oocyte growth, the total PGK-1 enzyme activity increases 40–50 fold and the pattern of PGK-1 isozyme expression changes giving an electrophoretic pattern now skewed in favour of the PGK-1B isozymic form. Activity of the PGK-1B isozyme exceeds that of PGK-1A in all growing oocytes with a total activity greater than 0·075 nmol h−1oocyte−1. Mice homozygous for eitherPgk-1allele show similar PGK-1 specific activities in somatic tissues where oneX-chromosome is active, but oocytes ofPgk-lbhomozygotes show a higher specific activity compared to those ofPgk-1ahomozygotes. Thus increased activity of the PGK-1B isozyme relative to the PGK-1A isozyme is specific to growing oocytes.

Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168.

Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis.

Is high mobility group protein 17 phosphorylated [formula omitted]? re-examination of the HeLa cell cycle data

When in vivo [ 32P] phosphate labeled HMG proteins from unsynchronized HeLa cells are separated by electrophoresis in acid-urea polyacrylamide gels, as opposed to separation in SDS-polyacrylamide, HMG 17 does not show any 32P incorporation. Likewise, no 32P radioactivity was found in HMG 17 protein isolated at different stages of the cell cycle from synchronized cells. By contrast, HMG 14 reveals a previously reported (Bhorjee, J.S. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6944–6948) cell cycle stage-specific dependent phosphorylation with maximum 32P radioactivity in the G 2 phase relative to G 1. Furthermore, HMG 14 is resolved into multiple electrophoretic forms as phosphoprotein in the acid-urea system. The results presented seriously question the data on the in vivo phosphorylation of HMG 17, and suggest that these be reevaluated.

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Properties, purification, and subunit structure of class III RNA polymerases.

The class III RNA polymerases present in Xenopus laevis tissues were solubilized and partially purified by ion exchange chromatography. Single chromatographic species were detected in extracts from ovary, liver, and a kidney-derived cultured cell, whereas two chromatographic forms were detected in liver extracts. All class III enzymes analyzed exhibited characteristic responses to ionic strength changes, synthetic polynucleotide templates, and alpha-amanitin. These chromatographic and catalytic properties readily distinguished all the class III enzymes from the corresponding class I and II enzymes but clearly failed to reveal any significant differences between the various ovarian and somatic class III enzymes (with the possible exception of one of the liver enzyme forms). The ovarian RNA polymerase III was completely soluble in low ionic strength buffers and was subjected to further purification via standard procedures involving differential centrifugation, ammonium sulfate fractionation, and ion exchange chromatography. Polyacrylamide gel electrophoresis under denaturing conditions revealed at least 10 distinct polypeptides (designated subunits a-j) which were consistently present in near equimolar amounts and which ranged in size from 155,000 to 19,000 daltons. Several smaller polypeptides (less than 19,000) were also evident in all preparations but were not further characterized. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms, although subsequent analysis on denaturing gels failed to reveal any differences in polypeptide composition. From the present data, it is estimated that there are about 4 X 10(9) molecules of RNA polymerase III per oocyte nucleus. This extraordinary level of soluble RNA polymerase III in part explains the ability of oocyte nuclei (or extracts) to support the active transcription of exogenous tRNA and 5 S RNA genes. The present and previous data concerning the properties and structures of the oocyte versus somatic cell enzymes also suggest that the RNA polymerase III present in the oocyte is functionally equivalent to a somatic cell enzyme and support the previous suggestion that this enzyme is conserved and used later in (somatic) embryonic cells.

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

Intraspecies Variability in the Esterases and Acid Phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of Unidentified Paramecia; Comparison with the P. aurelia Complex1

ABSTRACT. Enzyme electrophoresis was exploited to identify stocks of paramecia previously not identified to particular species. Stocks collected in India and one from Panama belong to Paramecium jenningsi, while others collected in Panama or in Brazil are assignable to syngen 2 of P. multimicronucleatum on the basis of similarity of their esterase and acid phosphatase phenotypes. Inclusion of these doubled the numbers of stocks available in the two species, thereby facilitating examination of intraspecies variation and comparison of particular features of intraspecies variation found for the P. aurelia complex. Variant stocks were observed in P. jenningsi and in syngens 2, 3, and 4 of P. multimicronucleatum. In some cases the variant lacked the enzyme; in others, a change in mobility of the enzyme occurred that resulted in an electrophoretic form similar to one common in another species. Unique phenotypes were displayed by the variants of syngen 2 in P. multimicronucleatum. Hypervariability for Esterase B was observed in this syngen, where, in addition, several subtypes were seen for three other esterases. Unique phenotypes and hypervariability were also noted in P. biaurelia. Clustered variations were observed in these species and in the P. aurelia species. Unlike the situation for members of the aurelia complex, where lack of geographical differentiation between stocks in the same species is a unique feature, some such differentiation does occur in P. multimicronucleatum‐2. The frequency of variant stocks in P. jenningsi was similar to that observed in the aurelia sibling species. In contrast, a significantly higher frequency of variant stocks was found in syngens 2, 3, and 4 of P. multimicronucleatum.

Reaction of coppersuperoxide dismutase with hydrogen peroxide: a possible source of Heterogeneity?

The reaction of hydrogen peroxide with copperzinc bovine superoxide dismutase at ratios of 0.2 to 4.0 per active site at pH 10.0 results in the formation of distinct electrophoretic forms of the enzyme. The mobilities of these forms are identical to the mobilities of the forms present in heterogeneous, untreated native samples in two different electrophoretic systems, pH 6.96 (TRIS-acetic acid) and pH 8.38 (TRIS-glycine) (Fig.1.). Increasing ratios of hydrogen peroxide result in an apparent increase in this heterogeneity in addition to progressive inactivation (Fig. 2). Inactivation of the dismutase and loss of histidine was reported by Hodgson and Fridovich [1]. Furthermore this reaction results in the loss of copper from the dismutase at all ratios of added peroxide and the loss of zinc at the higher ratios (Table I). The loss of copper parallels the increase in the faster moving species suggesting the possibility that this species is copperdeficient. However, in the pH 6.96 electrophoretic system, the mobility of this faster form does not coincide with the mobility of a copper-deficient form prepared by reconstituting the apoenzyme with equimolar copper and zinc [2]. If it is copper-deficient, the faster electrophoretic form must be modified by the reaction with peroxide. It should be noted that this form is not capable of giving rise to native enzyme upon the addiction of copper and zinc. In addition the reaction with peroxide forms distinct electrophoretic forms of the enzyme at the more physiological pH 7.0. Analysis of several purified preparations of superoxide dismutase have indicated that a ▪ ▪ lower copper content is correlated with an increased heterogeneity (Table II). For all samples listed but one, this heterogeneity was not altered by the addition of copper and zinc. In view of these observations we would like to suggest that the reaction with hydrogen peroxide in vivo may be the source of the heterogeneity so often seen in purified preparations of the copperzinc enzyme. Hydrogen peroxide is the product of the enzyme-catalyzed dismutation reaction of superoxide anion and its reaction with the dismutase, though slow, could lead to the accumulation of altered proteins t001 Loss of Copper and Zinc from Superoxide Dismutase in the Reaction with Hydrogen Peroxide at pH 10.0 H 2O 2 Copper Zinc site Enzyme Enzyme 0.0 1.30 1.92 0.49 1.23 1.94 0.98 1.18 1.69 1.46 1.16 1.79 1.96 1.00 1.23 2.96 0.87 3.96 0.85 1.08 t002 Copper Content and Heterogeneity in Preparations of Bovine Superoxide Dismutase Sample Copper %I %II %III Enzyme Sigma [1] 1.89 83 15 2 DDI 1.83 83 16 1 Sigma [2] 1.75 66 29 5 DDI 1.61 71 24 5 Malta 1.42 40 58 2 Sigma [2] dialyzed 1.30 52 34 14 that copurify with the native enzyme in classical isolation procedures.

Purification and characterization of esterase 6A, a trimeric esterase of the house mouse (Mus musculus).

Esterase 6A was isolated from mouse lung and purified 440-fold by ion-exchange chromatography, inverse ammonium sulphate gradient solubilization, gel filtration and isoelectric focusing. The resultant product was apparently homogenous by the criteria of polyacrylamide gel electrophoresis and immunodiffusion, and consisted of the electrophoretic form 6A3. A single species of subunit was present on sodium dodecyl sulphate gel electrophoresis. The molecular weight of the native protein was found to be about 178,000 with a subunit molecular weight of about 60,000. The equivalent weight obtained by active-site titration with diethyl-p-nitrophenyl phosphate was approximately 178,000 g/mol, indicating a functional asymmetry in the trimer. The enzyme was shown to have a high affinity for 4-nitrophenyl hexanoate (Michaelis constant Km = 4.4 mumol/l) with a relatively low catalytic efficiency (catalytic constant kcat = 12 s-1). Esterase 6A was immunologically related to esterase 1 and esterase 9, with which it is genetically closely linked. Further properties of the three esterases were compared.

Characterization of the Different Electrophoretic Forms of the Cadang-Cadang Viroid

SUMMARY The relationship between symptom development and changes in the fast and slow electrophoretic forms of the two cadang-cadang disease-associated RNAs (ccRNA-1 and ccRNA-2) has been examined. The fast form of each is present in trees for up to 2 years before the appearance of symptoms, indicating an incubation period of about 2 years. Trees showing the first symptoms (on the nuts) contain only the fast form of the ccRNAs; the development of leaf symptoms coincides with the first detection of the slow form. After this time, both slow and fast forms are found in the newly developing fronds for the next 9 to 12 months but then, as symptoms develop, new fronds contain only the slow form. Therefore, at later stages of disease, only the slow form can be detected in all fronds. The fast form of ccRNA-1 and/or ccRNA-2 therefore appears to be the infectious form in nature. The number of nucleotides in the four ccRNAs as determined by polyacrylamide gel electrophoresis under denaturing conditions are: ccRNA-1 fast, 250; ccRNA-1 slow, 300; ccRNA-2 fast, 500; ccRNA-2 slow, 600. On the basis of two-dimensional fingerprints of ribonuclease A and T1 digests of the four ccRNAs, it was concluded that the ccRNAs are composed of repeated sequences of ccRNA-1 fast, and each of the ccRNA-2 forms is a dimer of the respective ccRNA-1 form.

Regulation of Blood Oxygen Affinity in the Australian Blackfish Gadopsis Marmoratus

ABSTRACT The effects of thermal acclimation on whole blood oxygen affinity were examined in the freshwater blackfish Gadopsis marmoratus. Oxygen equilibrium curves for 20 °C-acclimated fish were shifted to the right of curves obtained for 10 °C-acclimated fish when determined at both 20 °C and 10 °C. Oxygen equilibrium curves obtained for solutions of stripped haemoglobin prepared from blood of 20 °C- and 10 °C-acclimated fish did not show the differences observed for whole blood. Thermal acclimation did not alter the number, migration rates, or relative amounts of the five electrophoretic forms of haemoglobin present in blackfish blood. The intraerythrocytic concentration of nucleoside triphosphates was higher in the 20 °C-acclimated fish than in 10 °C-acclimated fish, while the whole blood haemoglobin concentration was lower in the 20 °C-acclimated fish. These differences gave NTP : Hb4 molar ratios of 1·68 for the 20 °C-acclimated fish and 1·32 for 10 °C-acclimated fish. The effects of nucleoside triphosphates on oxygen affinity were similar for stripped haemoglobins of both acclimation groups. The change in NTP:Hb4 molar ratios with acclimation temperature acts to enhance oxygen unloading to the tissues rather than oxygen uptake at the gills at the higher acclimation temperature. As the waters inhabited by the blackfish retain high oxygen tensions at 20 °C, these changes in blood oxygen affinity could be considered adaptive if they were associated with elevated rates of oxygen-dependent metabolism at the higher temperatures.