Bovine Ribonuclease Research Articles

Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans → cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (τ = 500s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10°C. However, it is strongly accelerated (τ = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0°C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.

Distribution of two urinary ribonuclease-like enzymes in human organs and body fluids.

In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino acid sequence of snapping turtle (Chelydra serpentina) ribonuclease.

Snapping turtle (Chelydra serpentina) ribonuclease was isolated from pancreatic tissue. Turtle ribonuclease binds much more weakly to the affinity chromatography matrix used than mammalian ribonucleases. The amino acid sequence was determined from overlapping peptides obtained from three different digests. The N-terminal amino acid sequence of the protein determined by others [E. A. Barnard, M. S. Cohen, M. H. Gold J.-K. Kim (1972) Nature (Lond.) 240, 395-398] and homology were used as additional evidence for several overlaps. The polypeptide chain consists of 119 amino acid residues. Compared to most ribonucleases the N-terminal residue, three residues in the loop near residue 71 and two residues in the loop near residue 114 are deleted, and there is one additional residue in the loop near residue 23. The half-cystines at positions 65 and 72, which form a disulfide bond in mammalian ribonucleases, are not present in turtle ribonuclease. Turtle ribonuclease differs from bovine ribonuclease at 70 of the 118 positions where both proteins have amino acid residues. Turtle ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Ala-Ser (positions 76-78).

Prediction of sequential antigenic regions in proteins

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.

Insulin degradation: radioimmunoassay for glutathione-insulin transhydrogenase and its application.

A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [125I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunoassay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human gamma-globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620 +/- 80 versus 1070 +/- 30 fmol/l, p less than 0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue and lowest in the muscle.

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids. Antipeptide antibody Ribonuclease Secondary structure Immunization

Purification and Properties of Bovine Kidney Ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.

A method for determining oligo- and poly(ADP-ribosy)ated enzymes and proteins in vitro

A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed. This method is based on the facts that in Mg 2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg 2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated. When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg 2+-depleted conditions but their activity was unchanged. Under Mg 2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation. When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity. As used here successfully, the Mg 2+-depleted and Mg 2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.

Association of newly synthesized islet prohormones with intracellular membranes.

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

Relationships between nonhyperbolic kinetics and dimeric structure in ribonucleases.

Dimers of bovine pancreatic RNase A give nonhyperbolic saturation curves for the substrate of the second, rate-limiting step of the reaction. Under the same conditions, the monomeric native enzyme shows Michaelis-Menten kinetics. Naturally dimeric bovine seminal RNase, which has been found to give nonhyperbolic saturation curves, loses this property upon monomerization. It is proposed that when RNase monomers are arranged in a quaternary structure, they assume a conformation which enables them to be modulated in their catalytic activities. A correlation is suggested between this effect and the quaternary structure proposed for both of these dimeric ribonucleases, in which composite active sites are generated by the mutual exchange of the NH2-terminal ends of the two monomers.

Immunohistochemical demonstration of ribonuclease and amylase in normal and neoplastic parotid glands

Antibodies specific for bovine ribonuclease A (antiRNase A) were raised in rabbits, and immunologic cross-reactivity between bovine RNase A and human salivary gland RNase was demonstrated. The antiRNase A served as the primary antibody in the peroxidase-antiperoxidase immunohistochemical technique. Paraffin blocks of five normal human parotids and 20 parotid tumors were examined. In normal parotid and in cases of cystadenoma lymphomatosum, immunoreactive RNase was localized in the ductal epithelium, evidence of the ductal cell origin of these benign tumors. RNase immunoreactivity was noted in the adenomatous structures and in cells isolated in the myxoid matrix of pleomorphic adenomas, which supports recent evidence of an epithelial origin of these tumors. Malignant acinar cells of acinic cell carcinoma were strongly positive for immunoreactive RNase, while acinar cells of normal parotid were uniformly negative. This expression of the gene for RNase A probably represents a loss of differentiation (i.e., control) of the neoplastic acinar cells. Further evidence for this hypothesis was obtained by treating these tumors with an antihuman salivary amylase antibody, which is localized in normal acinar cells. No immunoreactive amylase was observed. The results support the idea that immunoreactivity need not accompany enzyme activity, as the presence of immunoreactive RNase was noted in all neoplastic tissues examined. Immunohistochemical localization of two antigens in the same tissue demonstrates the varied biochemical changes associated with parotid neoplasia.

Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 2—characteristics of positive ionization spectra of peptides,m/z 858 tom/z 5729

Sixteen peptides ranging in molecular weights from 858 to 5729 were examined under positive ionization fast atom bombardment conditions employing a high field magnet mass spectrometer and data system. The contributions of the polyisotopic elements to the molecular protonated ion envelope become important in the interpretation of the data at higher mass. For masses less than 2000 u, the most abundant ion within this envelope is the monoisotopic molecular protonated ion. Above 2000 u, the most abundant ion in the envelope is a polyisotopic molecular protonated ion. Characterization of the peptide requires identification of both the molecular protonated ion envelope and significant fragment ions. Partial spectra displaying both the molecular ion and significant fragment ions are presented for mastoparan (mol. wt = 1478), somatostatin (mol. wt = 1637), bovine parathyroid hormone (1–34) (mol. wt = 4106), and bovine insulin (mol. wt = 5729). A partial spectrum for bovine ribonuclease A (mol. wt = 13673) displayed significant fragment ions that identified the protein. The types of fragment ions included those that indicated the amino acid sequence and the location of disulfide bonds. The abundance of these ions appears to be influenced by the characteristics of the noble gas fast atom beam and the sample matrix.

Metallocenes for protein modification: II. Maleylirontetracarbonyl-maleyl-ribonuclease

Reaction of bovine ribonuclease A with maleic anhydride in the presence of the inhibitor, pyrophosphate, results in maleyl-ribonuclease, which is fully active after removal of the pyrophosphate. Further reaction of this derivative with maleic anhydride irontetracarbonyl, a metallocene-like compound, results in the formation of maleylirontetracarbonyl-maleyl-ribonuclease, which is rich in metallo spectral properties. This product is largely inactive and contains close to 1 mole of the maleylirontetracarbonyl group per mole of enzyme. Acid hydrolysis and amino acid analysis of this derivative, RNase, and maleyl ribonuclease, each reacted with 1-fluoro-2,4-dinitrobenzene, show that the matallocene-like moiety modifies lysine(s) of maleylribonuclease. The corresponding 14C-maleylirontetracarbonyl-maleyl-ribonuclease is then prepared and reduced with mercaptoethanol in the presence of urea, alkylated with iodoacetamide, and digested with trypsin plus chymotrypsin.The maleyl peptides and maleylirontetracarbonyl peptides are isolated by affinity chromatography using an appropriate antibody-Sepharose conjugate. A 14C-peptide is purified by subsequent ion-exchange chromatography and recovered in a 55% absolute overall yield. This peptide comprises residues 40–46. Since lysine 41 is the only lysyl residue in this peptide, at least 55% of the maleylirontetracarbonyl group is attached to lysine 41.

5S rRNA gene transcription factor IIIA alters the helical configuration of DNA.

Relaxation of Xenopus 5S plasmid DNA (pX1o8) in the presence of transcription factor (TF) IIIA reduces the linking number of the DNA. Parallel experiments with plasmid pMB9 or cloned hepatitis B viral DNA indicate a degree of non-specific unwinding by TF; however, 60% of the effect observed for pX1o8 is due to specific interaction of TF IIIA with the 5S rRNA gene internal promoter sequence. The extent of unwinding (0.2-0.4 helical turn per TF IIIA binding site) is not consistent with the complete denaturation of the 50-base-pair TF binding site; however, it is consistent with a change in helix rotation, denaturation of 2-4 nucleotides per binding site, or DNA wrapping about a protein core. We show that proteins other than TF IIIA (bovine serum albumin and RNase) have no effect on the linking number of DNA when present during relaxation and that the unwinding activity associated with TF is heat labile. These results suggest that TF IIIA may facilitate transcription by altering the helical configuration of 5S DNA.

Dimerization of deoxyribonuclease I, lysozyme and papain. Effects of ionic strength on enzymic activity.

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskocil, J., and Libonati, M. (1980) Biochim. Biophys. Acta. 609, 40-52]. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease, hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength. Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate. The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one. 2. The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme. A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.

On the free radical-induced aggregation of ribonuclease - a pulse radiolysis study using the light scattering detection method.

The aggregation of bovine ribonuclease (RNase) induced by either .OH or Br(2) radicals has been studied. These radicals, which were generated by pulse radiolysis of aqueous solution with 16 MeV electrons, readily attack the enzyme thus producing RNase-radicals which subsequently combine. At initial radical concentrations low enough (less than 0.1 radical per enzyme molecule) to prevent multimerizations other than dimerization, rate constants for the latter process were determined from the increase of the light scattering intensity after the pulse: 2k2 = (22 +/- 0.3) 106 l/mol s (Br(2) initiated dimerization) and (5.4 +/- 0.4) 106 l/mol s (.OH initiated dimerization). The G-values for dimerization are: 1.3 (Br(2) and 0.85 (.OH). Transient optical absorption measurements revealed the existence of phenoxyl radicals of tyrosine (TyrO.) that decayed with a rate constant of 2 K2 (total) = (5.5 +/- 0.7) 106 l/mol s (Br(2)-case). The difference between k2 and k2 (total) presumably indicates the occurrence of disproportionation.

Why peptide synthesis continues to remain a formidable challenge

AbstractStriking advances have been made in the synthesis of biologically active peptides. The synthesis of fully active crystalline bovine ribonuclease A by Yajima and Fujii [(1981) Biopolymers 20, 1855–1863] represents an exciting milestone. The main factors contributing to this success include sequential coupling of many small segments and a sophisticated multistage protocol for the final acidolytic cleavage of all protecting groups. However, syntheses of large peptides and proteins and even of many medium‐sized peptides continue to present formidable challenges. In peptide coupling the challenge is to join large peptide segments efficiently and in good yields. For solid‐phase synthesis the challenge is application of polar supports and alternative Nα‐protection. With protecting groups the challenge is to achieve quantitive cleavage at the end of synthesis. Concerning racemization the challenge is to develop sensitive and rapid procedures for measuring racemization during coupling. For purification the challenge is to improve the existing procedures and to search for novel alternative separation principles.

Purification and characterization of human pancreatic ribonuclease.

A ribonuclease (RNase) has been isolated from normal human pancreas obtained upon autopsy. About 5 mg of RNase is normally recovered per kilogram of pancreas, equivalent to ca. 70% of the total activity and a 700-fold purification from the initial acidified extract. The specific activity of the purified enzyme is identical with that of bovine pancreatic ribonuclease, and a single component is found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and reversed-phase high-pressure liquid chromatography. Aggregation of the protein is found upon ultracentrifugation under native and denaturing conditions, and several bands of equal specific activity are seen in polyacrylamide gel electrophoresis of the native protein. At least two components are glycoproteins. A molecular weight of 15 000 is estimated from sodium dodecyl sulfate gel electrophoresis, gel filtration, and amino acid and peptide analyses. The enzyme is related to bovine pancreatic RNase, but distinguishable by amino acid analysis, tryptic peptide maps, and low cross-reactivity of antibodies with the heterologous enzymes. The human enzyme is also inactivated by treatment with iodoacetic acid at pH 5.5 and is essentially identical with bovine RNase in its far-ultraviolet circular dichroism spectrum. The human RNase is like bovine pancreatic RNase catalytically; RNA is cleaved at pyrimidine residues, and activity against poly(cytidylic acid) is high.

Bovine ribonucleases: Identification of tissue-specific enzymes by immunological methods

1. 1. Antibodies directed against bovine brain or bovine pancreatic ribonucleases (EC 3.1.4.22, ribonucleate 3′-pyrimidino-oligonucletidohydrolase) have been shown to be highly specific and sensitive in enzyme quantitation. In competitive binding radioimmunoassays, cross-reactivity with the heterologous ribonucleasesis less than 0.1% and sensitivity for detection of the homologous enzyme is 1–10 ng per assay, comparable to enzymatic analytic l procedures. 2. 2. Immunological analysis of each enzyme in crude extracts of brain and pancreas accurately measures the level of each enzyme. Radioimmunoassay also shows materials that cross-react with each antibody preparation in extracts of liver, kidney, and seminal vesicles, but in most tissues the level of immunological cross-reactivity is not sufficient to account for all enzymatic activity. Instead, these tissues have one or more RNases which are structurally different from either the brain or pancreatic enzymes. 3. 3. Gel filtration of extracts usually separates pancreatic-like and brain-like immunological cross- reactivity. Ribonuclease(s) which are not closely related to either the brain or pancreatic enzymes are sometimes apparent in the elution patterns. 4. 4. Cross-reactivaty of anti-bovine brain ribonuclease with extracts of pig, rat, chicken, or turtle brain is very low. It is suggested that structural evolution of the brain ribonucleases may have been rapid, as previously suggested for pancreatic ribonucleases.

Activating effect of several enzyme-proteins on the mutagenicity of amino-thiol compounds.

Amino-thiol compounds were found to show mutagenicity, when they were activated with S-9 Mix. Further, the mutagenicity also appeared when these amino-thiol compounds were activated with pepsin, catalase, D-amino acid oxidase and thermolysin. All of their activating effects remained even after they were heated in a boiling water bath for 10 min. On the other hand, pancreatic lipase, lysozyme, peroxidase, papain, bovine serum albumin, casein, hemoglobin, RNase, gelatin and ovoalbumin did not show any effect on the mutagenicity of these amino-thiol compounds. Kinetic studies of the reaction between these aminothiol compounds and pepsin or catalase suggested that the activation seems to be caused by proteins but not enzymatically.