Native Rabbit Research Articles

Structural studies on fructose-1,6-bisphosphatases from rabbit liver, kidney, and muscle

Fructose-1,6-bisphosphatases (EC 3.1.3.11) isolated from rabbit liver and kidney appear to have identical primary structures, as deduced from their tryptic peptide maps and the peptide patterns obtained after cleavage with cyanogen bromide and chromatography on Sephadex G75. The enzyme isolated from rabbit skeletal muscle, on the other hand, yields distinctly different fingerprints and cyanogen bromide cleavage products. The results indicate that animal cells possess two genes that code for fructose-bisphosphatase. Native rabbit liver fructose bisphosphatase contains a single tryptophan located near the NH 2-terminus, and the NH 2 terminal-BrCN peptide containing this residue has been identified in the Sephadex G75 filtrates.

Allotypic Suppression in Rabbits: Competition for Target Cell Receptors between Isologous and Heterologous Antibody and between Native Antibody and Antibody Fragments

Neonatal injection with various foreign proteins (normal goat serum, human Cohn fraction II, human albumin) caused a stimulation of immunoglobulin synthesis. This effect was not antigen-specific and did not constitute a conventional antibody response directed against the injected substance. When this stimulatory effect of foreign protein was minimized, the heterologous (goat) anti-rabbit allotype antibody and rabbit antibody F(ab') 2 fragments not only failed to induce suppression but also competed with the suppression-inducing native rabbit antibody. Allotypic suppression in a rabbit can thus only be induced by an antibody molecule possessing an intact Fc portion of isologous, rabbit origin. Antibody to Ae14, an allotypic specificity located on the Fc portion, failed to induce suppression or stimulation of immunoglobulin synthesis. This was attributed to the position of Ae14 in the cell membrane which reduced its accessibility to antibody.

Studies on the structure and activity of rabbit Clq (a subcomponent of the first component of complement).

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15-18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52 degrees C (but not at 49 degrees C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

Transformation of neutral to alkaline fructose 1,6-bisphosphatase: Converting enzyme activity in the large-particle fraction from rabbit liver

A liver particle fraction containing lysosomes catalyzes the conversion of native rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11), having a neutral pH optimum, to a modified form with an alkaline pH optimum. The “converting enzyme” activity is partially recovered with the membranes from disrupted particles, and is also detected in “intact” particles isolated and maintained in isotonic buffered sucrose. The converting enzyme activity associated with the membrane fraction is expressed at pH 6.5, but not at pH 4.5, although activity at the lower pH appears when the enzyme is released from the membranes with Triton X-100. In contrast, proteolytic activity as measured with peptide and protein substrates is maximal at pH 5.0 or below, and is the same for the membrane-bound or solubilized proteases. The results suggest that a specific converting enzyme, at least partially associated with a particle (possibly lysosomal) membrane, is responsible for the modification of fructose bisphosphatase and the change in its catalytic properties.

Specificity of rheumatoid factors cross-reacting with human and rabbit IgG.

The reaction of rheumatoid factors with rabbit IgG sensitized erythrocytes was inhibited by Fc fragments and by a mixture of Facb and pFc' fragments of rabbit IgG, but not by either of the latter two fragments alone. The reaction was also inhibited by heat aggregated human IgG1 and IgG2 myeloma proteins, depending on the amount of aggregates present, but irrespective of their Gm types. No inhibition was seen with IgG3 or IgG4 proteins. Immunosorbent studies showed that the antigen of human IgG involved in these reactions is probably closely related to the γ1–2‐4 antigen, which is a major human IgG antigen interacting with rheumatoid factors. Quantitative haemagglutination inhibition studies in an Auto‐Analyzer showed that native human IgG very weakly inhibited the reaction of rheumatoid sera with rabbit IgG. Isolated aggregated human IgG inhibited far better, and gave an inhibition curve indicating the formation in aggregated human IgG of new antigens very similar to antigens of native rabbit IgG. Thus, in rheumatoid arthritis there may be a humoral immune response also against antigens present in aggregated but not in native human IgG, giving the cross‐reaction with native rabbit IgG.

Changes in sulfhydryl groups of honeybee glyceraldehyde phosphate dehydrogenase associated with generation of the intermediate plateau in its saturation kinetics.

Honeybee and rabbit muscle GPDH were studied to obtain information at the chemical level regarding anomolous saturation kinetics of the honeybee enzyme. Results demonstrate that the enzyme's sulfhydryl groups are implicated in the process. Measured by DTNB titration, native honeybee GPDH has one less active SH than the native rabbit muscle enzyme and displays changes in overall sulfhydryl reactivity after preincubation with G-3-P or G-3-P plus NAD+. The total DTNB reactive sulfhydryls of rabbit muscle GPDH are not changed by preincubation with NAD+ or G-3-P; honeybee GPDH, under certain conductions of preincubation with these ligands, shows a decrease of two total DTNB reactive SH groups. This difference has been confirmed by an independent experiment in which the two enzymes were carboxymethylated with C-14 bromoacetic acid.

Heat-labile accessory factor involved in vaccinia virus plaque neutralization.

A heat-labile accessory factor present in native (unheated) normal serum was found to replace the vaccinia virus neutralizing activity of immune serum destroyed by heating at 56° C for 30 minutes. Treatment of serum samples with ammonium hydroxide or cobra snake venom did not significantly reduce the activity of immune serum. Also, inactivation studies with the properdin deficient (PR) fraction of immune serum after zymosam treatment did not alter the neutralizing activity of immune serum. The neutralization kinetics of vaccinia virus were altered in the presence of the accessory factor. However, the optimum conditions for neutralization were not changed. In the hemagglutination-inhibition (HI) test procedure, heating decreased the HI activity of vaccinia immune serum, but the addition of native normal rabbit serum did not restore the lost activity. Although individual components of complement might affect vaccinia neutralization, it appeared that a heat-labile accessory factor, other than properdin or complement, was required for the complete neutralizing activity of vaccinia immune serum.

An investigation of the subunit structure and AMP-deaminases from rabbit and chicken muscle

Native rabbit and chicken muscle AMP-deaminases have been compared by gel filtration chromatography. These studies, along with previously-reported ultracentrifugal analyses, suggest that these enzymes are virtually identical in size and shape. Chemical and physical evidence is presented which shows that: (a) each enzyme has four subunits; (b) the molecular weights of these subunits are indistinguishable (69,000 Daltons and 73,000 Daltons, as determined by gel filtration chromatography in guanidine hydrochloride and electrophoresis in SDS-containing polyacrylamide gels, respectively; and (c) the native enzymes are not stabilized by disulfiede bridges. Although previous studies suggest that these enzymes share certain structural features, the results presented here provide direct evidence, at the molecular level, that the adenylate deaminases from these sources have similar quaternary structures.

The Nucleotide and Acyl Group Content of Native Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase

Abstract Glyceraldehyde 3-phosphate dehydrogenase (GP-dehydrogenase) isolated from rabbit muscle by the procedure of Ferdinand (Biochem. J., 92, 578 (1964)), has two previously unreported properties. 1. The coenzyme-binding sites usually filled with NAD+ in native GP-dehydrogenase contain instead the NAD+ degradation product, ADP-ribose. 2. Several of the four active sites contain covalently bound 3-phosphoglyceroyl group. The enzyme isolated by the Ferdinand procedure is in reality the acyl enzyme intermediate in the physiological oxidative phosphorylation of glyceraldehyde 3-phosphate. The occurrence of ADP-ribose appears to be a consequence of the isolation procedure in vitro, but the recovery of acyl enzyme suggests that GP-dehydrogenase is in an acylated state in vivo. The data required to estimate the sarcoplasmic concentrations of NAD+, NADH, the glycolytic intermediates, and the glycolytic enzymes are available in the literature. These estimates and the known effect of acylation on the GP-dehydrogenase-NAD+ affinity suggest two physiological regulatory roles for acyl-GP-dehydrogenase in muscle. One function is the regulation of coenzyme activities and oxidation-reduction potential as the affinity of enzyme for NAD+ changes with degree of acylation, itself dependent on the metabolic state of the tissue. The other is the buffering by acyl enzyme of the more dilute glycolytic intermediates during rapid changes in metabolic state. Although these roles must be considered hypothetical, the finding in skeletal muscle of levels of acylated GP-dehydrogenase much higher than those of many glycolytic intermediates should lead to revision of the accepted glycolytic pathway to include 3-phosphoglyceroyl-GP-dehydrogenase as a significant metabolite.

Reversible Dissociation of Tetrameric 7.4 S Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase into 4.4 S Dimers by Ammonium Sulfate and into 3.2 S Monomers by KCl

Abstract At 0°, native tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (7.4 S) undergoes a reversible inactivation and dissociation into 4.4 S dimers or 3.2 S monomers, depending upon the species of salt present and upon the protein concentration. These dissociation processes have been studied using sucrose density gradient centrifugation. With 0.10 mg per ml of enzyme in 0.15 m ammonium sulfate, 4.4 S dimers predominate; with 0.05 mg per ml of enzyme in 0.15 m KCl, 3.2 S monomers predominate. At other protein concentrations in the two systems, mixtures are produced. KCl gives greater dissociation at all protein concentrations. It also destabilizes at high salt concentrations (1.0 to 2.5 m). In contrast, destabilization in ammonium sulfate is maximal at 0.15 m; 0.5 and 1.0 m ammonium sulfate stabilize against dissociation essentially completely. The native enzyme in the absence of salts shows very slow, very slight dissociation into dimers. Inactivation and dissociation do not occur at 23° in 0.15 m ammonium sulfate and only slightly in 0.15 m KCl, and then only at very low protein concentrations. Inactivation and dissociation are reversed by warming to 23° and greater than 80% of the activity is recovered. The reversible dissociation and inactivation occur at both pH 7.0 (0.1 m imidazole) and at pH 9.5 (0.2 m glycine). A reducing agent (0.1 m β-mercaptoethanol) has to be present for activity to be recovered. These data prove that this enzyme at 0° exists as an equilibrium mixture which favors association to tetramers at high protein concentrations and dissociation into dimers or monomers or both at low protein concentrations. The dimers and monomers appear to be fairly compact.

Specificity of Rabbit Antibody to Formaldehyde Treated Rabbit Serum Albumin

Formaldehyde is a common cause of allergic contact dermatitis in man, but unlike many sensitizing agents, it is probably too small to be a hapten. Formaldehyde reacts with the primary amino, groups of proteins to form methylol groups or methylene bridges. Similar chemical substitutions are known to create new antigenic determinants in proteins by changing the net charge and native conformation. This study was designed to explore the mechanism of formaldehyde hypersensitivity. Native rabbit serum albumin altered by reactivity with formaldehyde-C 14 was immunogenic in the rabbit. Antisera reacted with a specific fraction of the immunizing antigen obtained by cationic exchange chromatography. The moles of formaldehyde taken up by this fraction were less than the number of primary amino groups blocked, indicating that methylene bridges were formed within or between molecules. The antiserum to formaldehyde-altered RSA did not precipitate with native RSA, acetylated RSA or other formaldehyde-altered proteins. Antisera prepared to acetylated RSA did not react with formaldehyde-altered RSA. These data suggest that these antibodies are primarily directed toward a new conformation of albumin produced by methylene bridges.

X-ray modification of the catalytic and allosteric functions of fructose-1,6-diphosphatase

1. 1. Native rabbit liver fructose-1,6-diphosphatase ( d-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11) and enzyme stimulated by disulfide formation have been irradiated in solution with X-rays. 2. 2. The dose-response curve for the native enzyme showed an initial stimulation of catalytic activity, followed by subsequent exponential inactivation. The disulfide-modified enzyme was inactivated exponentially with no initial stimulation. The G values for inactivation of catalytic activity were 0.017 and 0.009 for the native and the disulfide-modified enzymes, respectively. 3. 3. The allosteric activity of the native enzyme, as measured by the inhibition by AMP, was much more radioresistant than the catalytic activity. In the modified enzyme, these two functions were of comparable radiosensitivity. 4. 4. The ability of the enzymes to be stimulated by mixed disulfide formation was about 10 times as sensitive to X-ray inactivation as was the catalytic function. The loss of sensitivity to stimulation by disulfides was due to destruction of 5–6 specific SH groups which were particularly radiosensitive ( G value, 1.2). 5. 5. The X-ray-induced reduction in enzyme activity was associated with destruction of enzyme SH groups of low radiosensitivity ( G value, 0.07. The loss of enzyme activity was partly reversed by addition of cysteamine after exposure. The results indicate that sulfhydryl groups play an important, but not exclusive role in the X-ray inactivation of fructose-1,6-diphosphatase. 6. 6. The presence of AMP during irradiation protected the enzyme almost completely against inactivation of the allosteric function. The results indicated that the X-ray inactivation of the allosteric function of fructose-1,6-diphosphatase is due to a specific modification of the binding sites for AMP.

Studies on the Structure and Function of Muscle Aldolase: IV. THE ACTION OF DILUTE ALKALI ON PRIMARY STRUCTURE AND ITS EFFECT ON THE DETERMINATION OF SUBUNIT MOLECULAR WEIGHT

Abstract In 0.05 m potassium phosphate buffer (pH 7.0, 20°), native rabbit muscle aldolase has been found to exhibit a molecular weight (Mn ∼ Mw ∼ Mz) close to 160,000, in agreement with previous findings. Upon exposure to cold alkaline borate buffer (pH 12.5, µ = 0.17, 0°), the enzyme spontaneously dissociates into its monomers which subsequently undergo slow hydrolytic degradation. Compensating for electrostatic charge effects and extrapolating the data to zero time in alkali, a subunit molecular weight of 41,400 to 42,000 is obtained. If the native enzyme is exposed to alkaline conditions at 20°, the subunits undergo an initial rapid degradation which eventually yields alkali-resistant material. Similar results are obtained with succinyl aldolase subunits under equivalent conditions. Acrylamide gel electrophoresis of alkali-treated succinyl aldolase (pH 12.5, 30°) results in the appearance of a limited number of electrophoretic bands. This finding suggests that specific alkali-labile peptide bonds are involved in the degradation process. As a further consequence of the action of alkali, native aldolase shows increased solubility properties. Both native and succinyl aldolase manifest new NH2-terminal serine, threonine, and glycine residues after treatment with borate buffer at pH 12.5 and 30°. Prolonged exposure to alkali results in increased liberation of the above end groups, but no evidence is obtained for other NH2-terminal residues except for a small amount of proline.

The Behavior of Autologous Thyroglobulin in the Circulation of Rabbits Immunized with Either Heterologous or Altered Homologous Thyroglobulin

Summary Injection of adequate doses of NaI131 into iodine deficient but otherwise normal rabbits results in disruption of thyroid structure with release of I131 thyroglobulin into the circulation. In these rabbits, the I131 thyroglobulin is eliminated at an exponential rate with a half-life of 2.3 days. The release of autologous I131 thyroglobulin in rabbits previously immunized with either arsanil-sulfanil-thyrogobulin (homologous) or a heterologous thyroglobulin causes a secondary stimulation resulting in an immune elimination of the I131 thyroglobulin and production of antibody to native rabbit thyroglobulin. Complexes formed between the circulating I131 thyroglobulin and the newly synthesized antibody deposit along the glomerular basement membrane of the kidney resulting in mild glomerular inflammatory changes and proteinuria.

Studies of incomplete antibodies. II. Interactions with rheumatoid factors and complement.

AbstractNonagglutinating human (anti‐Rh) and rabbit (anti‐G and anti‐g) iso‐antibodies and agglutinating rabbit and hamster γG antibodies against sheep erythrocytes were tested for rheumatoid factor reactivity and for complement‐fixation using papain‐treated and nontreated cells. Although the papain treatment increased the agglutination of the erythrocytes by the antibodies, no change was observed in the complement‐fixing properties. The minimum amount of rabbit γG antibody on the cell surface required for the RF reactivity was the same with papain‐treated and non‐treated cells and of the same magnitude in three serological systems (rabbit incomplete anti‐G and anti‐g and rabbit antisheep cell). This amount of sensitizing antibody was 1/8–1/16 of the amount required for complement‐fixation and for agglutination by a type of RF reactivity that could not be inhibited by native rabbit γG globulin.

汚染指標菌としての腸球菌について

1. The most probable numbers (MPN) of Escherichia coil and Enterococcus in feces were determined in 10 head each of 9 animal species, including man, by the A. Y. I.-E. A. C. broth method. E. coli was found in dog, man, sheep, Japanese native ox, chicken, goat, guinea pig, rabbit, and dairy cow in the decreasing order of MPN. Enterococcus was detected in dog, sheep, chicken, man, goat, rabbit, guinea pig, Japanese native oxen, and dairy cow in the same order as above.2. The number of enterococci was about 1/20 to 1/10 that of E. coli organisms in all the animals, except the chicken, in which it was almost equal to the latter.3. When 487 samples of food were examined, 20.5 per cent of them was positive for E. coil and 13.7 per cent for Enterococcus. The resultant difference was significant at 1 per cent of a when examined by the X2-test.4. The isolated organisms were classified into Streptococcus faecium (41.0%), Str. faecalis (36.4%), Str. durans (5.36%), Str. faecalis var. liquefaciens (4.6%), and atypical strains (12.64%).5. Str. faecium was mostly found among the strains isolated from feces and Str. faecalis among those isolated from food.6. The differences between the two bacterial species in the numbers of organisms isolated from “soft cream” and fresh milk were not significant at 5 per cent of α when examined by t-distribution.These results seem to indicate that detection of enterococci, -when performed at the same time with that of E. coli organisms, may be useful as additional means for the determination of the degree of food contamination.

Kinetic Properties of Native and Carboxypeptidase-altered Rabbit Muscle Aldolase

Kinetic Properties of Native and Carboxypeptidase-altered Rabbit Muscle Aldolase

Distribution of Cottontail Rabbits in Washington State

The Provisional List of the Land Mammals of the State of Washington (Taylor and Shaw, 1929) gives a rough outline of the range of the native cottontail rabbit. During the past several years a number of records have been obtained that make it possible to show the distribution of this mammal in the State with considerable accuracy. Since the list was published at least four successful plantings of cottontail rabbits from the eastern part of the United States also have been made. In view of these facts it is thought advisable to review the status of the genus Sylvilagus in Washington.'