EXPLANATION OF THE OBSERVATION OF PANCREATIC RIBONUCLEASE ACTIVITY AT pH 4.5

A recent conclusion that beef pancreas contained a molecular species of ribonuclease with intrinsically high activity at pH 4.5 has been found to be incorrect. The particular assay used in the earlier experiments gives anomalous results at acid pH in the presence of low concentrations of ions such as phosphate which was used during the fractionation. By turning to the more widely employed form of the perchloric acid precipitation assay, interference is avoided and the ribonuclease in beef pancreas is confirmed as consisting almost completely of the molecular species well-characterized as ribonuclease A. The clarification of the assay question permits a clear interpretation of the results of each step of the chromatographic purification procedure that led to the initial conclusion, including an artifact that arose when gel filtration was attempted with distilled water rather than with buffer.     

MECHANISM OF GLUTATHIONE REGENERATION OF REDUCED PANCREATIC RIBONUCLEASE A

A comparative study was made of the kinetics of glutathione regeneration of reduced pancreatic ribonuclease A, as determined by circular dichroism, sulfhydryl oxidation and the kinetics of reactivation. Four sulfhydryls were reoxidized prior to any large circular dichroic changes or recovery of enzymatic activity. The helical and β segments in ribonuclease were shown to reform at approximately the same rate. The results are discussed in terms of a regeneration mechanism for ribonuclease involving (1) nucleation, (2) polypeptide backbone refolding, and (3) reshuffling of incorrectly paired disulfide bonds.     

THE PRIMARY STRUCTURE OF MUSKRAT PANCREATIC RIBONUCLEASE

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32–33) and Ser-Arg (tentative position 75–76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23–25 and 99–102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31–42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44–52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn- Val-Thr (62–64).     

STUDIES ON THE PRIMARY STRUCTURE OF BISON PANCREATIC RIBONUCLEASE

Bison pancreatic ribonuclease was isolated by affinity chromatography. Thermolysin and tryptic digestion of denaturated protein, and subtilisin digestion of native protein yielded peptides, which were purified and submitted to amino acid analysis. These peptides, together with partial sequence data obtained by Stewart & Stevenson (16) overlap the entire amino acid sequence of bison ribonuclease. No differences with bovine ribonuclease were found, although there may be differences in state of amidation of some residues.     

REACTION OF BOVINE PANCREATIC RIBONUCLEASE A WITH 6-CHLOROPURINE RIBOSIDE 5‘-MONOPHOSPHATE

The n.m.r. spectra of native S-peptide and of S-peptide II, a derivative obtained after reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5′-monophosphate, both in D₂O and in urea-d₄, were obtained with a 270 MHz Fourier transform spectrometer. From these spectra it was possible to assign most of the proton resonances of the peptide and the position of the labelling group, the α-NH₂ of Lys-1, was also deduced.     

MODIFICATION OF THE PROPERTIES OF BOVINE PANCREATIC RIBONUCLEASE A BY COVALENT ATTACHMENT OF D-GLUCONYL-GLYCINE RESIDUES*

Bovine pancreatic ribonuclease A was reacted with D-gluconyl-glycine azide in aqueous solution at pH 8.9, in absence of phosphates. Five out of 11 amino groups can be reproducibly modified and the penta D-gluconyl-glycinated ribonuclease A had greater than 70% of the enzymic activity of the unmodified enzyme toward cytidine 2′, 3′-cyclic phosphate as well as uridine 2′, 3′-cyclic phosphate and yeast RNA. The kinetic parameters K_m and k₂ of the modified enzyme were calculated from double-reciprocal Lineweaver-Burk plots, using cytidine 2, 3′-cyclic phosphate as the substrate. The native and chemically modified protein exhibited identity in their reversible thermal transitions at neutral pH, with midpoint at about 60°. Circular dichroism measurements indicated that the overall conformation of the D-gluconyl-glycinated enzyme is not significantly different from that of the unglycosylated parent enzyme. The modified protein was less sensitive than the native ribonuclease A to attack by chymotrypsin, pepsin and elastase, indicating a protecting effect of the D-gluconyl-glycine units. Similar properties are shown by the glycosylated bovine pancreatic ribonuclease B.     

A SYNTHETIC STUDY OF THE EFFECT OF TYROSINE AT POSITION 120 OF RIBONUCLEASE

[Tyr¹²⁰]-RNase 111–124 and [Ala¹²⁰]-RNase 111–124 were synthesized and purified to chromatographic and electrophoretic homogeneity. The peptides were mixed noncovalently with RNase 1–118 that had been prepared by enzymatic degradation of native bovine pancreatic ribonuclease A. The activities of the resulting complexes were compared with that of the corresponding complex containing the natural peptide sequence, [Phe¹²⁰]-RNase 111–124, and with native RNase, using yeast RNA, C > p and U > p as substrates. The Tyr¹²⁰ and Phe¹²⁰ complexes were equally active against RNA and C > p, but the Tyr¹²⁰ complex was twice as active as the Phe¹²⁰ complex against U > p. The complex with alanine at position 120 was only 1% as active toward C > p as the complex containing phenylalanine. The prediction that giraffe RNase, which contains tyrosine at position 120, would be relatively more active toward U > p than C > p compared with bovine RNase was verified.     

MODIFICATION OF BOVINE PANCREATIC RIBONUCLEASE A WITH PYRIDOXAL 5‘-PHOSPHATE. ISOLATION AND IDENTIFICATION OF DERIVATIVES

Bovine pancreatic ribonuclease A was allowed to react with pyridoxal 5′-phosphate at pH 8 and 4°. After reduction with sodium borohydride, the principal products formed in the initial stages of modification were separated by successive chromatography on CM-cellulose and SP-Sephadex. The isolated derivatives were identified as Nα-(P-pyridoxyl)-Lys-1-, Ne-(P-pyridoxyl)-Lys-7-, and Ne-(P-pyridoxyl)-Lys-41-ribonuclease A. These results are interpreted in terms of the specificity of pyridoxal-P as a protein reagent.     

A SQUARE PYRAMIDAL MODEL FOR RIBONUCLEASE ACTION

A mechanism incorporating a square pyramidal model (SP) is presented for the action of bovine pancreatic ribonuclease. Its formulation is based on structural principles governing pentacoordinate behavior. The model is compared with a previous trigonal bipyramidal (TP) representation with regard to the geometry of the active site and enzyme constraints. Of two variants of the SP model, an adjacent (cis displacement) and in-line (trans displacement) process, the in-line mechanism, as with the TP model, fits existing model studies. Consideration of the energetics of the SP vs. the TP model leads to an estimated energy difference of about 1–2 kcal/mol. This suggests that the preferred model may be intermediate in geometry between the two idealized representations for the enzymatic hydrolysis. Comparisons are made showing that pseudorotation is an unlikely process in either model.     

CHEMICAL MODIFICATION OF METHIONINES OF RIBONUCLEASE A WITH o-BENZOQUINONE

The accessibility of methionines in RNAase A to reaction with OBQ has been studied at highly acidic pH. The differences between the rate constants of reactions of the methionine and methionines of RNAase A with OBQ is a reflection on the limited accessibility of methionines in the protein conformation. Nevertheless, at sufficiently high OBQ concentration, all the four methionines of the enzyme can be modified. At lower concentration of OBQ, a derivative may be prepared in which a specific methionine is modified. The introduced chromophore ionizes at around pH 3 in this derivative. The derivative has partial activity towards RNA which is enhanced on addition of S-protein.     

PROTEOLYTIC SUSCEPTIBILITY AND METHIONINE MODIFICATION OF MONODEAMIDATED RIBONUCLEASE A

The susceptibility of a monodeamidated RNAaseA (RNAaseAa₁) towards carboxypeptidaseA, α-chymotrypsin and pepsin has been studied. Similar to RNAaseA, the C-terminal of RNAaseAa₁ is not available for carboxypeptidase A hydrolysis. The thermal stability of RNAaseAa₁ as probed through chymotryptic digestion is found to be less than that of RNAaseA. Preliminary chromatographic analysis of the digested material, however, suggests that the nature of thermal transition might be the same in the two proteins. Pepsin inactivates RNAaseAa₁ more slowly than does RNAaseA. Accordingly, less peptide bonds, almost half that of RNAaseA, are cleaved by pepsin in RNAaseAa_1. The accumulation of RNAase-P type intermediates is not evident during peptic digestion of RNAaseAa_1. Reaction with O-benzoquinone at low pH shows that methionines of the deamidated protein seem to have higher reactivities. These observations indicate a different structure for RNAaseAa₁ at elevated temperature and low pH.     

SYNTHESIS OF ANALOGS OF THE N-TERMINAL EICOSAPEPTIDE SEQUENCE OF RIBONUCLEASE A

For a better understanding of the role played by glut amine 11 either in the catalytic function of ribonuclease A or in the S-peptide/S-protein association process the following S-peptide analogs have been synthesized: [Orn¹⁰,Leu¹¹]- and [Orn¹⁰, Lys¹¹]- S peptide. The S-protein activating ability of the two synthetic S-peptide analogs was tested, before and after guanidination, by exploring their capacity to generate ribonuclease activity, against RNA as substrate, when recombined with S-protein.     

NUCLEAR MAGNETIC RESONANCE STUDY OF A HYBRID OF BOVINE AND RAT RIBONUCLEASE

A hybrid RNase S', consisting of synthetic S-peptide of rat ribonuclease and bovine S-protein, was studied by ¹H n.m.r. to determine the effects of the many N-terminal amino acid replacements in rat S-peptide as compared to bovine S-peptide. As judged from the aromatic resonances, the conformation of the hybrid RNase S' is essentially identical to the conformation of bovine RNase S'. Notably, the active-site histidines 12 and 119 were not affected by the substitutions in the S-peptide, confirming earlier findings that the catalytic properties of naturally occurring RNases are modulated by the S-protein part of the molecule only. However, the resonances of Tyr-25 and His-48, which in RNase A are involved in a pH-dependent conformational transition, appeared to be different in the hybrid RNase, demonstrating that amino acid replacements may influence the structure of the protein locally.     

番麻蛋白酶活性中心基团的初步研究

在不引起酶变性的条件下,用半胱氨酸(Cys)专一性化学修饰试剂碘乙酸、对氯汞苯甲酸(?)(CMB),5,5′-二硫双硝基苯甲酸(DTNB)对酶分子进行化学修饰后,酶活性显著下降。发现酶的失活程度与修饰剂浓度间存在着化学计量关系,同时底物(酪蛋白)对酶活性有明显的保护作用。说明被修饰的部位(Cya残基)是酶的活性中心基团。Cys对酶有明显的激活作用;同时,(?)CMB、DTNB、碘乙酸对酶活性有明显的抑制作用(?)CMB抑制或O_2氧化而失活的酶溶液,又可被Cys重新激活,而恢复其活力,进一步证明其活性中心存在着Cys残基。以上事实均与典型的植物巯基蛋白酶——木瓜蛋白酶一致,说明番麻蛋白酶是一种巯基蛋白醇,其活性中心存在着Cys。     

SYNTHESIS OF PEPTIDE ANALOGS OF THE N-TERMINAL EICOSAPEPTIDE SEQUENCE OF RIBONUCLEASE A.

Synthesis of the S-peptide analog in which the arginyl residue in position 10 and the histidyl residue in position 12 are simultaneously replaced by two ornithines is described. The stereochemical homogeneity of the [Orn ¹⁰, Orn ¹²]-S-peptide has been assessed by digestion with aminopeptidase M. The ability of the synthetic S-peptide analog to bind and to activate S-protein was tested, before and after guanidination, by exploring its capacity to compete with S-peptide for S-protein and to generate ribonuclease activity when recombined with S-protein. The binding capacity of the synthetic material has been also evaluated by differential spectroscopy. Both the examined products failed to activate S-protein at a molar ratio as high as 500:1 with RNA, cytidine 2′, 3′-cyclic phosphate, and cytidylyl (3′-5′) cytidine as substrate. Moreover, inhibition studies and spectrophotometric measurements showed that the synthetic S-peptide analog as well as its guanidinated derivative are also unable to bind to S-protein. It seems that the histidyl residue in position 12 in the S-peptide sequence, in addition to the critical role played in the catalytic function, should be also important in the association process between S-peptide and S-protein.     

BOVINE SERUM ALBUMIN IN LITHIUM CHLORIDE SOLUTIONS. ASSOCIATION BEHAVIOR AT NEUTRAL pH

Measurements of light scattering at 546 nm were carried out on bovine serum albumin in LiCl solutions at neutral pH (ca. 5.2) and room temperature. The concentration of LiCl varied from 0.1 to 7.0 M. There was a relatively small increase in the molecular weight of the protein when the concentration of LiCl in aqueous solutions was in the range between 0.1 M and 3.0 M. A major increase occurred when the concentration of LiCl exceeded 3.0 M. Preferential binding of LiCl to the protein varied in the same direction: negative when the concentration of LiCl was below 3.0 M and positive when the concentration was above 3.0 M. A reaction pattern suggested that the aggregation is preceded by a reversible conformational change of the monomer and the aggregation is a combination of denatured monomers.     

Different AT1 receptor subtypes at pre- and postjunctional sites: AT1A versus AT1B receptors

Angiotensin (AT) II is known to enhance responses to electrical field stimulation (EFS) via AT1 receptors located on sympathetic nerve terminals. Differences in potency exist between AT1 receptor antagonists regarding the inhibition of the prejunctional and postjunctional AT1 receptors. It is hypothesized that prejunctional AT1 receptors might belong to the AT1B receptor subtype. Accordingly, the authors investigated whether AT1B receptor inhibition by high concentrations of PD123319 could suppress ATII-augmented noradrenergic transmission (prejunctional) in the rabbit thoracic aorta by means of a noradrenaline spillover model. Additionally, the influence of PD123319 on ATII-enhanced constrictor responses to electrical field stimulation was investigated in the isolated rabbit mesenteric artery. Furthermore, the authors investigated whether PD123319 could influence the constrictor responses (postjunctional) to ATII in both preparations. In the thoracic aorta, ATII (10 nM) caused a significant enhancement of EFS-evoked [3H]-noradrenaline release by a factor of 2.0 +/- 0.1. This reinforcement could be inhibited by PD123319 (0.1, 1, and 10 microM). The constrictor response to ATII was unaffected by PD123319. In the mesenteric artery, ATII (0.5 nM) caused a significant enhancement of constrictor responses to EFS by factors of 2.9 +/- 0.3, 2.3 +/- 0.3, and 1.6 +/- 0.1 at 1, 2, and 4 Hz, respectively. This enhancement could be attenuated by PD123319 (1 and 10 microM). The constrictor response to ATII was unaffected by PD123319. It is concluded that the prejunctional AT1 receptors belong to the AT1B subtype whereas postjunctional AT1 receptors do not.     

IN VITRO MEASUREMENT OF FIBER DISSOLUTION RATE RELEVANT TO BIOPERSISTENCE AT NEUTRAL pH: AN INTERLABORATORY ROUND ROBIN

Measurements are presented of the dissolution rates in neutral pH simulated lung (SLF) of several man-made vitreous fibers (MMVF) and crocidolite asbestos that were recently in chronic rodent inhalation studies. The measured dissolution rate depended strongly on the fiber composition. The MMVF tested dissolved from 30 times to nearly 1000 times faster than the crocidolite asbestos. Measurements were made in flow-through equipment in four different laboratories in North America and Europe. The standard deviations of the measured values for each fiber were typically between 30 and 50% of average value. It is believed that in order to be relevant to the dissolution of long fibers the extracellular fluid of the lung, the in vitro measurement must be performed at a rate high enough that corrosion products do not accumulate in sufficient concentration affect the dissolution rate.     

CRYSTAL AND MOLECULAR STRUCTURE OF A 4-OXIMINO-5-IMINO-PYRAZOLINE ACTIVE ESTER

The crystal structure of an active 4-oximino-5-imino-pyrazoline ester has been determined by single-crystal X-ray diffraction methods. The possible reasons for its high reactivity towards nucleophilic reagents are briefly discussed.