Search

Histamine intolerance is associated with impaired histamine metabolism due to diamine-oxidase (DAO) enzyme deficiency at intestinal level. The recommended strategy to alleviate the symptomatology is a low-histamine diet supplemented with DAO. Different sources of DAO enzyme have been characterized in the literature (animal, vegetal and microbial), although only the porcine kidney protein extract has been approved as food supplement. Despite optimistic clinical results of DAO supplementation, variability in enzymatic activity of this type of active ingredient is reported, potentially linked to manufacturing processes. The aim of this work was to evaluate the impact of the raw material and the technological process of obtaining the porcine kidney protein extract on DAO activity, and its evolution during storage at room temperature (25 °C) and refrigeration (4 °C). Twenty batches of DAO-containing extract were analyzed and found to have a consistent histamine-degrading activity. The powder extract was prepared by mincing and homogenizing porcine kidneys, defatting with acetone, and drying at 40 °C for 6 h. The inclusion of a biocidal step before acetone extraction was evaluated on DAO activity. The use of sodium hypochlorite and irradiation (8kGy) did not have a negative impact on DAO activity. Moreover, the inclusion of an initial freeze-drying step of the raw material yielded an extract with higher DAO activity. On the other hand, the effect of applying higher temperatures (70 °C, 80 °C, and 90 °C) during the extract drying process for 3 and 6 h was evaluated, reporting a significant decrease of the enzymatic activity, with losses of 10–20 % for every 10 °C increase in temperature. Refrigeration was the only storage method capable of preserving enzymatic activity for at least 24 months. The manufacturing and storage of porcine kidney extract are crucial steps in the formulation of DAO supplements suitable for the treatment of histamine intolerance.

Vasculitic peripheral neuropathy (VPN) is caused by vessel inflammation leading to peripheral nerve injury of acute-to-subacute onset. When VPN occurs in the context of systemic disease it is classified as Systemic Vasculitic Neuropathy (SVN) and as Non-Systemic Vasculitic Neuropathy (NSVN) when restricted to the nerves. This study aimed to compare the clinical characteristics, biopsy findings and disease outcome in patients with VPN. Clinical records of adult patients with VPN diagnosed at our institution between June-2002 and June-2019 were retrospectively reviewed. Demographic characteristics, clinical manifestations, nerve conduction studies, nerve biopsies, treatment and clinical evolution were analyzed in all patients with at least 6 months follow-up. Twenty-five patients with VPN were included (SVN, n = 10; NSVN, n = 15). No significant differences in demographic or clinical features were found between groups. The median delay between symptom onset and nerve biopsy was significantly longer in NSVN patients (10 vs 5.5 months, p = 0.009). Erythrocyte sedimentation rate (ESR) values over 20 mm/h were significantly more common in SVN patients (100% vs. 60%, p = 0.024). Nerve biopsies showed active lesions more frequently in treatment-naive patients compared to those who had received at least 2 weeks of corticosteroids (92% vs 38%; p = 0.03), with a higher proportion of definite VPN cases (92 vs 46%; p = 0.04). Although the clinical manifestations are similar, ESR is an important tool to help distinguish between both conditions. Early nerve biopsy in untreated patients increases diagnostic accuracy, avoiding misdiagnosis.

El estudio de las estructuras que conforman el plexo braquial se ha visto particularmente beneficiado con las imágenes de alta resolución que brindan los equipos de resonancia 3T. El plexo braquial puede presentar mononeuropatías o polineuropatías. Entre las primeras se distinguen los traumatismos, el atrapamiento, como el síndrome de la abertura torácica por costillas cervicales, apófisis transversas prominentes o tumores. En el grupo de las polineuropatías se encuentran los procesos inflamatorios, entre los que destacan la polineuropatía desmielinizante inflamatoria crónica, la plexitis autoinmunitaria (síndrome de Parsonage Turner), enfermedades granulomatosas y la neuropatía por radiación. Entre los procesos vasculares se mencionan la polineuropatía diabética y las vasculitis.

Ethics and communication with patients are basic transversal skills for the Degrees in Medicine, Physiotherapy and Nutrition. In this model, senior students use video to create simulated clinical cases with ethics and communication components, and conduct academic mentoring using these cases. To ensure quality video recordings, the students are introduced to visual language by students of the Master's in Communication, thus completing the learning cycle.

ABSTRACTThere is an urgent need to develop new agents against mycobacterial infections, such as tuberculosis and other respiratory tract or skin affections. In this study, we have tested two human antimicrobial RNases against mycobacteria. RNase 3, also called the eosinophil cationic protein, and RNase 7 are two small cationic proteins secreted by innate cells during host defense. Both proteins are induced upon infection displaying a wide range of antipathogen activities. In particular, they are released by leukocytes and epithelial cells, contributing to tissue protection. Here, the two RNases have been proven effective against Mycobacterium vaccaeat a low micromolar level. High bactericidal activity correlated with their bacterial membrane depolarization and permeabilization activities. Further analysis on both protein-derived peptides identified for RNase 3 an N-terminus fragment that is even more active than the parental protein. Also, a potent bacterial agglutinating activity was unique to RNase 3 and its derived peptide. The particular biophysical properties of the RNase 3 active peptide are envisaged as a suitable reference for the development of novel antimycobacterial drugs. The results support the contribution of secreted RNases to the host immune response against mycobacteria.

AbstractAntimicrobial peptides (AMPs) are nowadays understood as broad multifunctional tools of the innate immune system to fight microbial infections. In addition to its direct antimicrobial action, AMPs can modulate the host immune response by promoting or restraining the recruitment of cells and chemicals to the infection focus. Binding of AMPs to lipopolysaccharide is a critical step for both their antimicrobial action and their immunomodulatory properties. On the one hand, removal of Gram-negative bacteria by AMPs can be an effective strategy to prevent a worsened inflammatory response that may lead to septic shock. On the other hand, by neutralizing circulating endotoxins, AMPs can successfully reduce nitric oxide and tumor necrosis factor-α production, hence preventing severe tissue damage. Furthermore, AMPs can also interfere with the Toll-like receptor 4 recognition system, suppressing cytokine production and contributing to modulate the inflammatory response. Here, we review the immune system strategies devised by AMPs to avoid an exacerbated inflammatory response and thus prevent a fatal end to the host.Copyright © 2012 S. Karger AG, Basel

ABSTRACTAntimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. colimutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

Action stations: Antimicrobial peptides (AMPs) can be derived from amyloid‐prone regions by introduction of cationic residues at privileged positions (see picture). The design and testing of 24 de novo amyloid‐derived AMPs are described, and the likelihood of evolution of amyloid‐prone protein regions, devoid of antimicrobial activity, into potent antimicrobial domains is addressed.

Molekulare Evolution: Antimikrobielle Peptide (AMPs) können von amyloidanfälligen Regionen abgeleitet werden, indem man kationische Reste an privilegierten Positionen einführt (siehe Bild). 24 neue, von Amyloid abgeleitete AMPs wurden entworfen und getestet, und die Wahrscheinlichkeit für die Evolution von amyloidanfälligen Proteinregionen mit fehlender antimikrobieller Aktivität in potente antimikrobielle Domänen wurde untersucht.

Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp35as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34–38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal α-helix, the third loop, and the first and last β-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1–45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity.

Abstract Eosinophil cationic protein (ECP) is a ribonuclease secreted from activated eosinophils that may cause tissue injure as a result of eosinophilic inflammation. ECP possesses bactericidal, antiviral and helminthotoxic activity and inhibits mammalian cell growth. The mechanism by which ECP exerts its toxicity is not known but it has been related to the ability of the protein to destabilise lipid bilayers. We have assessed the involvement of some cationic and aromatic surface exposed residues of ECP in the inhibition of proliferation of mammalian cell lines. We have constructed ECP mutants for the selected residues and assessed their ability to prevent cell growth. Trp10 and Trp35 together with the adjacent stacking residue are critical for the damaging effect of ECP on mammalian cell lines. These residues are also crucial for the membrane disruption activity of ECP. Other exposed aromatic residues packed against arginines (Arg75-Phe76 and Arg121-Tyr122) and specific cationic amino acids (Arg101and Arg104) of ECP play a secondary role in the cell growth inhibition. This may be related to the ability of the protein to bind carbohydrates such as those found on the surface of mammalian cells.

Abstract The amino acid-decarboxylase activity of bacteria isolated from Engraulis encrasicholus preserved in ice was investigated throughout 23 days of storage. A total of 140 bacterial isolates were studied, including 37 enterobacteria, 23 pseudomonads, 49 Gram+ catalase+ cocci, 27 lactic acid bacteria and 4 enterococci. The percentage of strains that decarboxylated amino acids was low, 12% on average. None of the Gram+ catalase+ cocci showed aminogenic activity, but all enterococci isolates did. The enterobacteria with aminogenic capacity were identified as Enterobacter cloacae and they simultaneously produced putrescine and cadaverine (up to 500 mg/l), but not histamine. Among pseudomonads, two species decarboxylated ornithine (producing 350–650 mg/l putrescine): Pseudomonas cepacia and Pseudomonas fluorescens. Lactic acid bacteria decarboxylated tyrosine, yielding up to 2,000 mg/l tyramine, and they were identified as Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceous and Enterococcus spp. As a general rule, the positive Gram-negative isolates (enterobacteria and pseudomonads) were diamine producers and they were found only during the first 2 weeks of storage. In contrast, lactic acid bacteria and enterococci were mainly tyramine producers and they were isolated only at the end of storage.

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.

We examined the amino acid-decarboxylase activity of bacteria isolated throughout the ice storage of Mediterranean hake. Pseudomonas was the main microbial group in the fresh samples. Its number increased by up to 2 logarithmic units during the storage and 20% of the isolates showed the ability to produce putrescine. Shewanella putrefaciens was the most prolific organism and became the dominant microbial group on day 6 of storage. It also had the highest proportion of aminogenic isolates and the strongest decarboxylase potential as a tyramine- and cadaverine-producing microorganism. A competitive procedure in biogenic amine production seems to have occurred when trimethylamine oxide was available in the culture medium. The abundance of Enterobacteriaceae increased gradually throughout the storage but never reached high counts and their isolates produced small amounts of cadaverine only. Although the counts of micrococci and staphylococci were considerably high, only two isolates were ornithine-decarboxylase positive. Neither lactic acid bacteria nor enterococci were positive.

The effect of delayed gutting on biogenic amine formation during ripening of European anchovies (Engraulis encrasicholus) was studied. The main difference between the two processes was the use of gutted or ungutted fish in the first step of ripening. Biogenic amines and microbial counts were studied in seven batches of samples taken during manufacturing from fresh anchovies and from anchovies throughout ripening. The formation of amines commonly related to fish spoilage (histamine, tyramine, cadaverine, putrescine and agmatine) was significantly higher in the first step of ripening of batches in ungutted fish. In contrast, no differences were observed between the content of the natural polyamines spermine and spermidine.

Efficient energy storage is one of the biggest problems facing alternative energy technologies. In whatever form the energy is stored, an alternative energy system usually requires a storage buffer between carrying energy input and the varying energy demand regime at the output end of the system. A method of energy storage is the use of the latent heat from Phase Change Materials (PCMs), for example salt hydrates. In this paper we tested the corrosion resistance of five commercial metals (aluminum, brass, copper, steel and stainless steel) in contact with two salt hydrates, commonly used as PCM, with a melting temperature in the range of 48 to 58°C (sodium acetate trihydrate and sodium thiosulfate pentahydrate) in experiments with a duration up to 70 days. The results demonstrated that brass and copper should be avoided when sodium acetate trihydrate is used in long term applications, but aluminum, steel and stainless steel can be used without problem. When the salt hydrate used is sodium thiosulfate pentahydrate, brass and copper should not be used in any case, aluminum and stainless steel can be used, and steel in contact with graphite should be monitored because corrosion could appear after some time of use.

We studied five patients with clinical and radiological evidence of syringobulbia (SB) to determine whether the distribution of lesions in relationship to the cardiorespiratory control networks in the medullary intermediate reticular zone (IRt) correlates with the presence of abnormalities in autonomic cardiovascular and respiratory control in these patients. All patients underwent high resolution MRI to characterize the size, volume and distribution of the SB lesions, cardiovascular autonomic function testing and polysomnography. One patient with bilateral IRt involvement at both the rostral and caudal medulla had orthostatic hypotension (OH), absent HRDB, abnormal Valsalva ratio, exaggerated fall of BP during phase II and absent phase IV during VM, and a dramatic fall of BP during head up tilt; this patient also had severe obstructive sleep apnea (OSA) and exhibited BP drops during each respiratory effort. A second patient, with bilateral IRt involvement restricted to the caudal medulla, had less severe cardiovascular autonomic dysfunction but also exhibited severe OSA. The other three patients had small SB cavities sparing the IRt and had sleep apnea but no autonomic dysfunction. Autonomic dysfunction could not be related to the size of the syrinx or the degree of atrophy in the cervical spinal cord in any of the five patients. Bilateral involvement of the IRt by SB produces cardiovascular autonomic failure and sleep apnea. In patients with more restricted lesions, autonomic and respiratory dysfunction may be dissociated. Clinico-radiological correlations using high resolution MRI assessment of medullary lesions can provide insight into the central organization of cardiovascular and respiratory control in humans.

The eosinophil cationic protein (ECP) is a basic secretion proteininvolved in the immune response system. ECP levels in biological fluidsare an indicator of eosinophil‐specific activation and degranulationand are currently used for the clinical monitoring and diagnosis ofinflammatory disorders. A polyclonal epitope‐specific antibody has beenobtained by immunizing rabbits with a conjugated synthetic peptide. Asequence corresponding to a large exposed loop in the human ECPthree‐dimensional structure (D115–Y122) was selected as a putativeantigenic epitope. The antibody was purified on an affinity columnusing recombinant ECP (rECP) as antigen. The antibody (D112–P123 Ab)specifically recognizes rECP and its native glycosylated andnonglycosylated forms in plasma, granulocytes, and sputum. The antibodydetects as little as 1 ng of rECP, can be used both in reducing andnonreducing conditions, and does not cross‐react with the highlyhomologous eosinophil‐derived neurotoxin or other proteins of thepancreatic ribonuclease superfamily.

The stability of the water-soluble B vitamins and the fat-soluble vitamins A and E of three liquid infant milks was followed throughout 12 months of storage at 20, 30 and 37 °C. Two samples were ‘follow-on’ milks from two different manufacturers and the other a ‘junior’ milk from one of these. The levels of water-soluble vitamins and vitamin E remained constant during storage at the three temperatures. In contrast, vitamin A decreased in samples from one manufacturer at all storage temperatures, and in samples from the other only at 37 °C. A kinetic equation was derived that made it possible to predict the influence of storage temperature on the shelf life of these kinds of products, together with the storage period during which levels of vitamin A recommended as adequate would remain.

To determine the frequency and types of abnormalities of respiratory control during sleep in syringomyelia and syringobulbia and to provide a basis to predict patients at risk of sudden death.

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1sites, located upstream of the P-O-5′ cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

To describe different types of involuntary movements and abnormal spontaneous electromyographic (EMG) activity in patients with syringomyelia.

Abstract.: The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p1 ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p2 and p0 have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p2 , and Lys-66 in p0 ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p2 are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p0 electrostatic interaction contributes only to binding of the RNA.

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p) to 3'-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA. There is much evidence that several subsites, in addition to the main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Parés, X., Nogués, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 abolishes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism.

The kinetics of the hydrolysis of cytidine 2′,3′-cyclic phosphate (C>p) to 3′-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3′-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2binding subsite are used. 2) Thekcat/Kmvalues with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcatobtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

The preparation of immobilized bovine pancreatic ribonuclease by covalent attachment to Sepharose 4B, with and without a spacer arm, is described. The coupling reaction was carried out at two different pH values, 8.5 and 10.5, and the different kinetic properties shown by the resulting preparations probably reflect the influence of the particular amino acid side-chains involved in the covalent coupling of the enzyme to the insoluble matrix. The strength of binding of mononucleotides, at 4°C, as deduced from the salt concentration at which they are eluted from an immobilized RNAase column, follows the order 5′-GMP > 5′-AMP > 3′-UMP > 3′-CMP. When binary mixtures of a 3′-pyrimidine nucleotide and a 5′-purine nucleotide are chromatographed jointly, a co-operative effect is found and the elution of either or both ligands is retarded. This behaviour can be explained in terms of the preferential binding of each kind of nucleotide to different sub-sites in the enzyme. The stoichiometry and association constant for 3′-CMP and 5′-AMP at pH 7.0 were also determined.