Primary preputial reconstruction following surgical excision of cutaneous mast cell tumours without penile amputation in eight dogs.

CASE HISTORY: Medical records from a single referral hospital (Davies Veterinary Specialists, Hitchin, UK) were reviewed to identify dogs (n = 8) with preputial cutaneous mast cell tumours (CMCT) that underwent surgical excision and primary preputial reconstruction, preserving the penis and urethra, after clients declined alternatives such as penile amputation and urethrostomy, from June 2017-June 2022. CLINICAL FINDINGS: Tumours had a median diameter of 21.5 (min 15, max 30) mm, were located cranioventrally (3/8), caudoventrally (1/8), laterally (2/8) and dorsally (2/8) relative to the prepuce and were diagnosed as CMCT based on cytology. No dogs had hepatic or splenic metastasis on cytology but inguinal lymph node metastasis was identified in 3/4 dogs sampled. TREATMENT AND OUTCOME: The owners of all dogs had declined penile amputation and scrotal urethrostomy. The CMCT were excised and primary reconstruction of the prepuce performed. Surgical lateral margins of 10, 20 or 30 mm were used and the deep margin excised the inner preputial lamina or underlying muscular fascia. The deep margin for caudoventral CMCT involved excision of the underlying SC adipose tissue. Preputial advancement was performed in 3/8 dogs to achieve adequate penile coverage. Histopathology confirmed all CMCT were Kiupel low grade, Patnaik grade II with complete margins in 6/8 dogs but identified metastasis only in one inguinal lymph node from one dog. Two dogs encountered minor complications (infection and a minor dehiscence) and one dog had a major complication (infection with major dehiscence). Median follow-up duration was 125 weeks, excluding one dog with 4 weeks of follow-up. None of the dogs experienced local recurrence or died of mast cell disease during the available follow-up period. CLINICAL RELEVANCE:  This clinical study evaluated a surgical alternative to penile amputation and advanced reconstructive techniques for Kuipel low/Patnaik grade II preputial CMCT when these procedures were declined by owners. Surgical excision of preputial CMCT with lateral margins of 10, 20 or 30 mm with primary preputial reconstruction is achievable with low morbidity and a good outcome when penile amputation and scrotal urethrostomy is not an option.

CT features of cutaneous and subcutaneous canine mast cell tumors and utility of conventional and indirect lymphography to detect clinically unknown mast cell tumors and to map the sentinel lymph nodes.

Computed tomography is frequently used to stage canine mast cell tumors (MCTs). The aims of this prospective, observational study were to describe the CT features of MCTs, to evaluate the performance of CT in detecting additional or incidental MCTs, to distinguish between cutaneous (cMCT) or subcutaneous (scMCT) MCTs, and to identify one or multiple sentinel lymph nodes (SLNs) by indirect CT lymphography (ICTL). Seventy-two dogs affected by 111 MCTs were included. The recorded parameters were: shape, size, attenuation (Hounsfield units [HU]), location (cutaneous or subcutaneous), and presence of fat stranding. The SLNs were compared with the regional lymph nodes and supplementary MCTs were registered. Mast cell tumors mostly appeared with well-defined margins (89%), round/oval shape (71%), homogeneous enhancement (90%) with a mean postcontrast density of 62.0 ± 23.4 HU and associated with fat stranding (43%). Cutaneous mast cell tumors were more frequently round (P = .003), whereas scMCTs were oval (P = .011) with a larger mean maximal diameter (2.91 ± 1.57 cm vs 1.46 ± 1.28 cm, P = .002) and more feeding vessels (77% vs 39% P = .044). Compared with histopathology, CT accuracy in differentiating cMCTs and sMCTs was 57%, with an interobserver agreement of 88% (three reviewers). Indirect CT lymphography showed the SLN in 82 of 85 (97%) cases, 32% of them not corresponding to the regional node. CT showed additional or incidental MCTs in 23 of 72 (32%) dogs. In conclusion, the common CT appearance of canine cMCTs and scMCTs is reported with some statistical differences between the two categories. CT is useful in identifying clinically undetected MCTs and SLNs, although it shows low accuracy in distinguishing between cMCT and scMCT.

SIRT6 deficiency in endothelial cells exacerbates oxidative stress by enhancing HIF1α accumulation and H3K9 acetylation at the Ero1α promoter.

BACKGROUND: SIRT6, an important NAD+ -dependent protein, protects endothelial cells from inflammatory and oxidative stress injuries. However, the role of SIRT6 in cardiac microvascular endothelial cells (CMECs) under ischemia-reperfusion injury (IRI) remains unclear. METHODS: The HUVECs model of oxygen-glucose deprivation/reperfusion (OGD/R) was established to simulate the endothelial IRI in vitro. Endoplasmic reticulum oxidase 1 alpha (Ero1α) mRNA and protein levels in SIRT6-overexpressing or SIRT6-knockdown cells were measured by qPCR and Western blotting. The levels of H2 O2 and mitochondrial reactive oxygen species (ROS) were detected to evaluate the status of oxidative stress. The effects of SIRT6 deficiency and Ero1α knockdown on cellular endoplasmic reticulum stress (ERS), inflammation, apoptosis and barrier function were detected by a series of molecular biological experiments and functional experiments in vitro. Chromatin immunoprecipitation, Western blotting, qPCR, and site-specific mutation experiments were used to examine the underlying molecular mechanisms. Furthermore, endothelial cell-specific Sirt6 knockout (ecSirt6-/- ) mice were subjected to cardiac ischemia-reperfusion surgery to investigate the effects of SIRT6 in CMECs in vivo. RESULTS: The expression of Ero1α was significantly upregulated in SIRT6-knockdown endothelial cells, and high Ero1α expression correlated with the accumulation of H2 O2 and mitochondrial ROS. In addition, SIRT6 deficiency increased ERS, inflammation, apoptosis and endothelial permeability, and these effects could be significantly attenuated by Ero1α knockdown. The deacetylase catalytic activity of SIRT6 was important in regulating Ero1α expression and these biological processes. Mechanistically, SIRT6 inhibited the enrichment of HIF1α and p300 at the Ero1α promoter through deacetylating H3K9, thereby antagonizing HIF1α/p300-mediated Ero1α expression. Compared with SIRT6-wild-type (SIRT6-WT) cells, cells expressing the SIRT6-H133Y-mutant and SIRT6-R65A-mutant exhibited increased Ero1α expression. Furthermore, ecSirt6-/- mice subjected to ischemia-reperfusion surgery exhibited increased Ero1α expression and ERS in CMECs and worsened injuries to microvascular barrier function and cardiac function. CONCLUSIONS: Our results revealed an epigenetic mechanism associated with SIRT6 and Ero1α expression and highlighted the therapeutic potential of targeting the SIRT6-HIF1α/p300-Ero1α axis.

Cutaneous mastocytosis in 8 young dogs and review of literature.

Cutaneous mastocytosis (CM) is a rare condition in young dogs characterized by multicentric cutaneous proliferation of neoplastic mast cells. Clinical data from 8 dogs that met inclusion criteria (age of onset less than 1.5 years, greater than 3 lesions) were obtained via a standardized survey. Biopsy samples were classified by the Kiupel/Patnaik grading systems and analyzed for c-KIT mutations. The median age of onset was 6 months (range: 2-17 months). Dogs had 5 to more than 50 lesions characterized as nodules, plaques, and papules. Seven dogs were pruritic. Clinical staging in 2 dogs did not reveal visceral involvement. No dogs had systemic illnesses at diagnosis. Histologically, CM was similar to cutaneous mast cell tumor (cMCT). Two dogs had neoplasms classified as high-grade/grade II while 6 dogs had low-grade/grade II neoplasms. No dogs had mutations in c-KIT exons 8 and 11. Treatment included antihistamines (8/8), corticosteroids (7/8), lokivetmab (3/8), and toceranib (1/8). Six dogs were alive with lesions at the end of the study with a median follow-up time of 898 days, while 2 dogs were euthanized. In dogs with high-grade/grade II neoplasms, one continued to develop lesions at 1922 days post-diagnosis, while the other dog was euthanized at 56 days post-diagnosis. One dog was euthanized 621 days post-diagnosis due to rupture of a neoplasm. CM occurs in young dogs and is histologically indistinguishable from cMCT. Current histologic grading systems did not apply uniformly to the dogs of the study and further studies are needed.

Expression of cannabinoid receptors CB1 and CB2 in canine cutaneous mast cell tumours.

Cannabinoid receptors (CB1 and CB2) belong to endocannabinoid system (ECS), which is also composed from endocannabinoids and the enzymatic systems involved in their biosynthesis and degradation. The expression of CB1 and CB2 have been previously identified in normal canine mast cell and in atopic dermatitis. Canine cutaneous mast cell tumours (cMCTs) are among the most common cutaneous neoplasms in dogs and have a highly variable clinical behaviour. Expression of CB1-CB2 was assessed by means of immunohistochemistry in thirty-seven dogs (from 2019 to 2021) with proven histological diagnosis of cMCT. Dogs were divided in two groups according to the Kiupel's grading system: high-grade (HG) cMCT and low-grade (LG) cMCT. A semiquantitative (score 0-3) and quantitative assessment of immunoreactivity (IR) was performed for each case. Our results show that there CB1 and CB2 are highly expressed in LG- cMCT, in contrast to HG- cMCT.

Plin5/p-Plin5 Guards Diabetic CMECs by Regulating FFAs Metabolism Bidirectionally.

BACKGROUND: Hyper-free fatty acidemia (HFFA) impairs cardiac capillaries, as well as type 2 diabetes mellitus (T2DM). Perilipin 5 (Plin5) maintains metabolic balance of free fatty acids (FFAs) in high oxidative tissues via the states of nonphosphorylation and phosphorylation. However, when facing to T2DM-HFFA, Plin5's role in cardiac microvascular endothelial cells (CMECs) is not defined. METHODS: In mice of WT or Plin5-/-, T2DM models were rendered by high-fat diet combined with intraperitoneal injection of streptozocin. CMECs isolated from left ventricles were incubated with high glucose (HG) and high FFAs (HFFAs). Plin5 phosphorylation was stimulated by isoproterenol. Plin5 expression was knocked down by small interfering RNA (siRNA). We determined cardiac function by small animal ultrasound, apoptotic rate by flow cytometry, microvessel quantity by immunohistochemistry, microvascular integrity by scanning electron microscopy, intracellular FFAs by spectrophotometry, lipid droplets (LDs) by Nile red staining, mRNAs by quantitative real-time polymerase chain reaction, proteins by western blots, nitric oxide (NO) and reactive oxygen species (ROS) by fluorescent dye staining and enzyme-linked immunosorbent assay kits. RESULTS: In CMECs, HFFAs aggravated cell injury induced by HG and activated Plin5 expression. In mice with T2DM-HFFA, Plin5 deficiency reduced number of cardiac capillaries, worsened structural incompleteness, and enhanced diastolic dysfunction. Moreover, in CMECs treated with HG-HFFAs, both ablation and phosphorylation of Plin5 reduced LDs content, increased intracellular FFAs, stimulated mitochondrial ß-oxidation, added ROS generation, and reduced the expression and activity of endothelial nitric oxide synthase (eNOS), eventually leading to increased apoptotic rate and decreased NO content, all of which were reversed by N-acetyl-L-cysteine. CONCLUSION: Plin5 preserves lipid balance and cell survival in diabetic CMECs by regulating FFAs metabolism bidirectionally via the states of nonphosphorylation and phosphorylation.

Ion/ion reactions with "onium" reagents: an approach for the gas-phase transfer of organic cations to multiply-charged anions.

The use of ion/ion reactions to effect gas-phase alkylation is demonstrated. Commonly used fixed-charge "onium" cations are well-suited for ion/ion reactions with multiply deprotonated analytes because of their tendency to form long-lived electrostatic complexes. Activation of these complexes results in an SN2 reaction that yields an alkylated anion with the loss of a neutral remnant of the reagent. This alkylation process forms the basis of a general method for alkylation of deprotonated analytes generated via electrospray, and is demonstrated on a variety of anionic sites. SN2 reactions of this nature are demonstrated empirically and characterized using density functional theory (DFT). This method for modification in the gas phase is extended to the transfer of larger and more complex R groups that can be used in later gas-phase synthesis steps. For example, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) is used to transfer a carbodiimide functionality to a peptide anion containing a carboxylic acid. Subsequent activation yields a selective reaction between the transferred carbodiimide group and a carboxylic acid, suggesting the carbodiimide functionality is retained through the transfer process. Many different R groups are transferable using this method, allowing for new possibilities for charge manipulation and derivatization in the gas phase.

Structural insights into group II intron catalysis and branch-site selection.

Group II self-splicing introns catalyze autoexcision from precursor RNA transcripts by a mechanism strikingly similar to that of the spliceosome, an RNA-protein assembly responsible for splicing together the protein-coding parts of most eukaryotic pre-mRNAs. Splicing in both cases initiates via nucleophilic attack at the 5' splice site by the 2' OH of a conserved intron adenosine residue, creating a branched (lariat) intermediate. Here, we describe the crystal structure at 3.0 A resolution of a 70-nucleotide RNA containing the catalytically essential domains 5 and 6 of the yeast ai5gamma group II self-splicing intron, revealing an unexpected two-nucleotide bulged structure around the branch-point adenosine in domain 6.

Catalytic site studies on tuna (Thunnus albacares) pyloric caeca aminopeptidase.

Tuna pyloric caeca aminopeptidase (tAP) is a glycosylated zinc-metalloenzyme containing apparently two identical subunits. The enzyme is reversibly inhibited in a time-dependent manner by amastatin. Slow development of tAP inhibition by this inhibitor could be demonstrated. Dissociation of the complex of tAP with amastatin is also slow. Two molar equivalents of the inhibitor are bound by the enzyme suggesting the presence of one catalytic site in each subunit. Chemical modification of tAP with 1-cyclohexyl-3-(2-morpholinoethyl) carbonyl-metho-p-toluene sulfonate and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone revealed the presence of essential acidic amino acid residues probably located at the active site. Compatible with the presence of arginine and tyrosine residues at the catalytic site of most metalloproteinases, tAP is reversibly inhibited by phenylglyoxal and inactivated by tetranitromethane in a time-dependent fashion. The rate of inhibition by these modifiers could be significantly decreased if the enzyme was previously treated with amastatin suggesting that the modified amino acid residues are located at the catalytic site. Diethylpyrocarbonate did not affect the activity of both native and zinc-depleted tAP suggesting that histidine is not involved in the zinc-ligand formation.

Interaction sites of ribosome-bound eukaryotic elongation factor 2 in 18S and 28S rRNA.

The involvement of ribosomal RNA in the binding of eukaryotic elongation factor eEF-2 to the ribosome was investigated. eEF-2 was complexed to empty reassociated 80S ribosomes in the presence of the nonhydrolyzable GTP analogue GuoPP[CH2]P. The formed complex was treated with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and micrococcus nuclease to allow specific modification at single-stranded regions of the rRNAs. The sites of modification were localized by primer extension using complementary deoxynucleotide primers and reverse transcriptase. The modification pattern was compared to that obtained from 80S ribosomes lacking bound eEF-2. Binding of the factor to the ribosome resulted in the protection of specific sites in both 18S and 28S rRNA, while the reactivity of 5.8S rRNA was unchanged. In 18S rRNA, the affected nucleotides were localized to the 5'- and 3'-domains, and in 28S rRNA the protected nucleotides were seen in domains II, IV, and V. The alpha-sarcin/ricin loop in domain VI of 28S rRNA was inaccessible for chemical modification even in the absence of bound eEF-2. However, the bound factor protected A4256, located in the alpha-sarcin/ricin loop, from ricin-induced depurination.

Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ.

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.

RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus.

We have examined the cis-acting RNA packaging signal (psi) from Moloney murine leukemia virus using a combination of chemical and primary sequence analysis techniques. For our chemical analyses, we used dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate as probes for RNA secondary structure. The structural information obtained from these studies was used to constrain computer algorithms for prediction of RNA secondary structure. In addition, we generated and analyzed a phylogenetic comparison of homologous sequences from related retroviruses. From these data, we have developed two models for the RNA secondary structure of the packaging signal psi. Both of these models suggest the presence of secondary structure elements in a region of the psi RNA known to be required for function.

Effect of carbodiimides on the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal membranes.

1. The hydrophobic N,N'-dicyclohexylcarbodiimide (DCCD) inhibits the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal fraction. 2. The inhibition is dependent on time and concentration, with half-maximal inhibition occurring at 0.4 mM concentration of carbodiimide after a 0.5 hr incubation at room temperature. 3. ATP does not protect against the inhibition. 4. Two water-soluble carbodiimides, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide (CMCD) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCD), are not inhibitory. 5. Inhibition of Mg(2+)-ATPase activity by DCCD is accompanied by covalent incorporation of the radioactive agent into the partially purified enzyme preparation.

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides.

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).

Structural and functional properties of the segments of lambda cro mRNA that interact with transcription termination factor Rho.

Termination of transcription at tR1, the Rho-dependent terminator between genes cro and cII of bacteriophage lambda, is dependent upon the structure of segments near the 3' end of the nascent cro gene transcript and on contacts between Rho protein and a 3' proximal segment called rut. The characteristics of the structure of cro RNA in the region from residue 220 to residue 355 in free, isolated RNA and in the presence of Rho or NusA proteins were analyzed by measuring relative rates of reactivity of individual nucleotides with chemicals and enzymes of defined specificities. The results indicate that the rut segments are single-stranded and become blocked to the action of the various probes in the presence of Rho factor. They also show that this region contains two stem-loop structures; one involves the boxB sequence of nutR, the other precedes the tR1 subsite II end points. The results provide direct evidence for a primary binding contact between Rho protein and the rut segment of cro RNA and demonstrate that this binding contact remains stable when the cro RNA is serving as a cofactor for ATP hydrolysis, an observation that is consistent with a mechanism in which Rho maintains contact with the rut region while it makes additional interactions with RNA that are coupled to ATP hydrolysis.

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

The action of a water-soluble carbodiimide on adenosine-5'-polyphosphates.

When aqueous solutions of adenosine-5'-mono-, di-, or triphosphates are treated with a water soluble carbodiimide the major product is the expected diadenosine-5'-5'-polyphosphate. The yields of these pyrophosphates are greatly increased in the presence of the Mg2+ ion. Adenosine-5'-tetraphosphate behaves differently. The major product is adenosine-5'-monophosphate. We believe that this hydrolysis occurs via a cyclic trimetaphosphate intermediate.

Papel del complemento en la recuperación funcional de los receptores para IgG bloqueador por complejos inmunes / Role of complement in the functional recovery of IgG receptors blocked by immune complexes

La interacción de complejos inmunes (CI) con receptores para el fragmento Fc de IgG (RFcgama) expresados en leucocitos pone en marcha mecanismos efectores y regulatorios de suma relevancia en el curso de la respuesta inmune. En trabajos anteriores, empleando la citotoxicidad celular dependiente de anticuerpos (ADCC) como expresión funcional de los RFcy, hemos demostrado que las células monocucleares periféricas humanas (CMPH), previamente bloqueadas en CI, recuperan la capacidad de mediar la ADCC a través de la activación de la vía alternativa del complemento (VAC). El objetivo de este trabajo fue analizar los mecanismos de recuperación funcional de los RFcgama cuando éstos han sido bloqueados por CI no fijadores de complemento (C). A tal fin, la IgG usada para preparar los CI se trató con carbodiimida (CDI), procedimiento que modifica su capacidad para fijar C, sin alterar mayormente su sitio de combinación con el antígeno, ni su habilidad para unirse a los RFcgama. Los resultados obtenidos demostraron que el C sólo es eficiente para revertir el bloqueo de la ADCC por CI, cuando éstos son capaces de fijar C

Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide.

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate.

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.