Prognostic role of the length of tumour-vein contact at the portal-superior mesenteric vein in patients having surgery for pancreatic cancer.

BACKGROUND: The length of tumour-vein contact between the portal-superior mesenteric vein (PV/SMV) and pancreatic head cancer, and its relationship to prognosis in patients undergoing pancreatic surgery, remains controversial. METHODS: Patients diagnosed with pancreatic head cancer who were eligible for pancreatoduodenectomy between October 2002 and December 2016 were analysed. The PV/SMV contact was assessed retrospectively on CT. Using the minimum P value approach based on overall survival after surgery, the optimal cut-off value for tumour-vein contact length was identified. RESULTS: Among 491 patients included, 462 underwent pancreatoduodenectomy for pancreatic head cancer. PV/SMV contact with the tumour was detected on preoperative CT in 248 patients (53·7 per cent). Overall survival of patients with PV/SMV contact exceeding 20 mm was significantly worse than that of patients with a contact length of 20 mm or less (median survival time (MST) 23·3 versus 39·3 months; P = 0·012). Multivariable analysis identified PV/SMV contact longer than 20 mm as an independent predictor of poor survival, whereas PV/SMV contact greater than 180° was not a predictive factor. Among patients with a PV/SMV contact length exceeding 20 mm on pretreatment CT, those receiving neoadjuvant therapy had significantly better overall survival than patients who had upfront surgery (MST not reached versus 21·6 months; P = 0·002). CONCLUSION: The length of PV/SMV contact predicts survival, and may be used to suggest a role for neoadjuvant therapy to improve prognosis.

ANTECEDENTES: El valor pronóstico de la longitud del contacto del tumor de la cabeza pancreática con las venas porta y mesentérica superior (portal-superior mesenteric vein, PV/SMV) en los pacientes sometidos a cirugía pancreática sigue siendo un tema controvertido. MÉTODOS: Se analizaron los pacientes diagnosticados de un cáncer de la cabeza pancreática a los que se realizó una duodenopancreatectomía cefálica entre octubre de 2002 y diciembre de 2016. El contacto tumoral con la PV/SMV se evaluó de forma retrospectiva mediante tomografía computarizada (TC). Se identificó el valor de corte óptimo para la longitud del contacto tumoral con la PV/SMV, utilizando el valor mínimo de la P basado en la supervivencia global (overall survival, OS) después de la cirugía. RESULTADOS: De 491 pacientes incluidos, en 462 pacientes se realizó una duodenopancreatectomía cefálica por cáncer de la cabeza de páncreas. En la TC preoperatoria, se detectó contacto tumoral con la PV/SMV en 248 (53,7%) pacientes. La OS de los pacientes en los que el contacto del tumor con la PV/SMV fue > 20 mm fue significativamente peor que en aquellos cuyo contacto fue ≤ 20 mm (mediana de supervivencia (median survival time, MST) 23,3 versus 39,3 meses; P = 0,012). En un análisis multivariado se identificó el contacto tumoral-PV/SMV > 20 mm como un factor independiente predictor de mala supervivencia, pero el contacto tumor-PV/SMV > 180° no fue un factor pronóstico. En los pacientes en los que el contacto tumor-PV/SMV fue > 20 mm en el TC preoperatorio, la OS en aquellos que recibieron tratamiento neoadyuvante fue significativamente mejor en comparación con los pacientes tratados directamente con cirugía (MST, no alcanzada versus 21,6 meses, P = 0,002). Conclusión La longitud del contacto tumoral con la PV/SMV predice la supervivencia, por lo cual dicha longitud podría jugar un papel en la indicación de tratamiento neoadyuvante para mejorar el pronóstico.

Major hepatectomy with combined vascular resection for perihilar cholangiocarcinoma.

BACKGROUND: Hepatectomy with vascular resection (VR) for perihilar cholangiocarcinoma (PHCC) is a challenging procedure. However, only a few reports on this procedure have been published and its clinical significance has not been fully evaluated. METHODS: Patients undergoing surgical resection for PHCC from 2002-2017 were studied. The surgical outcomes of VR and non-VR groups were compared. RESULTS: Some 238 patients were included. VR was performed in 85 patients. The resected vessels were hepatic artery alone (31 patients), portal vein alone (37 patients) or both (17 patients). The morbidity rates were almost the same in the VR (49.4 per cent) and non-VR (43.8 per cent) groups (P = 0.404). The mortality rates of VR (3.5 per cent) and non-VR (3.3 per cent) were also comparable (P > 0.999). The median survival time (MST) was 45 months in the non-VR group and 36 months in VR group (P = 0.124). Among patients in whom tumour involvement was suspected on preoperative imaging and whose carbohydrate antigen 19-9 (CA19-9) value was 37 U/ml or less, MST in the VR group was significantly longer than that in the non-VR group (50 versus 34 months, P = 0.017). In contrast, when the CA19-9 value was greater than 37 U/ml, MST of the VR and non-VR groups was comparable (28 versus 29 months, P = 0.520). CONCLUSION: Hepatectomy with VR for PHCC can be performed in a highly specialized hepatobiliary centre with equivalent short- and long-term outcomes to hepatectomy without VR.

Utility of remnant liver volume for predicting posthepatectomy liver failure after hepatectomy with extrahepatic bile duct resection.

BACKGROUND: Hepatectomy with extrahepatic bile duct resection is associated with a high risk of posthepatectomy liver failure (PHLF). However, the utility of the remnant liver volume (RLV) in cholangiocarcinoma has not been studied intensively. METHODS: Patients who underwent major hepatectomy with extrahepatic bile duct resection between 2002 and 2018 were reviewed. The RLV was divided by body surface area (BSA) to normalize individual physical differences. Risk factors for clinically relevant PHLF were evaluated with special reference to the RLV/BSA. RESULTS: A total of 289 patients were included. The optimal cut-off value for RLV/BSA was determined to be 300 ml/m2. Thirty-two patients (11.1 per cent) developed PHLF. PHLF was more frequent in patients with an RLV/BSA below 300 ml/m2 than in those with a value of 300 ml/m2 or greater: 19 of 87 (22 per cent) versus 13 of 202 (6.4 per cent) (P < 0.001). In multivariable analysis, RLV/BSA below 300 ml/m2 (P = 0.013), future liver remnant plasma clearance rate of indocyanine green less than 0.075 (P = 0.031), and serum albumin level below 3.5 g/dl (P = 0.015) were identified as independent risk factors for PHLF. Based on these risk factors, patients were classified into three subgroups with low (no factors), moderate (1-2 factors), and high (3 factors) risk of PHLF, with PHLF rates of 1.8, 14.8 and 63 per cent respectively (P < 0.001). CONCLUSION: An RLV/BSA of 300 ml/m2 is a simple predictor of PHLF in patients undergoing hepatectomy with extrahepatic bile duct resection.

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease.

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.

Crystallization of a new class of microbial ribonuclease from Rhizopus niveus.

Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid.

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.

Crystal structure of a plant ribonuclease, RNase LE.

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.

The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution.

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus.

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 A resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R-factor is 0.200 in the 10-2.5 A resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six alpha-helices and seven beta-strands, belonging to the alpha+beta type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site-directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.

Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase.

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.

Carboxamidomethylation of a ribonuclease from Aspergillus saitoi.

1) The inactivation of a RNase from Aspergillus saitoi (RNase Ms) was studied to obtain information on its active site. 2) Inactivation of RNase Ms by iodoacetamide was greater at an alkaline pH, and was protected more by 2',(3')-AMP than by 2',(3')-GMP. 3) Analysis of the hydrolysis products with 6 N HCl and alkaline treatment of carboxamidomethylated RNase Ms showed that the sites of reaction were one carboxyl group and one histidine residue. 4) Since the incorporation of a carboxamidomethyl group into carboxylic acid was not protected by 2',(3')-AMP, it was concluded that the formation of N1-carboxamidomethylhistidine was responsible for the loss of enzymatic activity of RNase Ms.

Further studies on the specificity of the minor ribonuclease from Aspergillus saitoi.

In order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25 degrees. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The Ki values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribonucleosides was in the order dT, dA, dG, and dC.

Carboxymethylation of a minor ribonuclease from Aspergillus saitoi.

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.

Primary structure of a minor ribonuclease from Aspergillus saitoi.

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.

Purification of acid ribonucleases from bovine spleen.

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.

The subsite structures of guanine-specific ribonucleases and a guanine-preferential ribonuclease. Cleavage of oligoinosinic acids and poly I.

In order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guanine-preferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI greater than p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n = 2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate.

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.

Isolation, characterization, and primary structure of a base non-specific and adenylic acid preferential ribonuclease with higher specific activity from Trichoderma viride.

In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Trv were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Trv towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Trv was determined by analyses of the peptides generated by digestion of reduced and carboxymethylated RNase Trv with Staphylococcus aureus V8 protease, lysylendopeptidase and alpha-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25,883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Trv and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.