Activity of two components of serum ribonuclease under conditions of physical exercise.

Serum ribonuclease activity before and after physical exercise in healthy persons was estimated. It is found that a work load of 6000 kgm/5 min increased ribonuclease activity measured at pH 8.5 and decreased the activity of the same enzyme measured at pH 7.0 in the presence of Zn SO4. The observed changes were more pronounced in untrained than in trained persons.     

Effect of exercise on ribonuclease activity in rat skeletal muscle.

Distribution of ribonuclease activity (measured at pH 7.6) in subcellular fractions of homogenates of rat skeletal muscle was investigated in sedentary animals and after 8 weeks running program. Training increased ribonuclease activity (expressed as units of enzyme per g of muscle protein). There was no increase in nuclear fraction, but in both cytoplasmic and mitochondrial fractions the RNA-ase activity increased 42% and 45% respectively.     

Effect of exercise on ribonuclease activity in rat skeletal muscle

Summary Distribution of ribonuclease activity (measured at pH 7.6) in subcellular fractions of homogenates of rat skeletal muscle was investigated in sedentary animals and after 8 weeks running program. Training increased ribonuclease activity (expressed as units of enzyme per g of muscle protein). There was no increase in nuclear fraction, but in both cytoplasmic and mitochondrial fractions the RNA-ase activity increased 42% and 45% respectively.Acknowledgments. I would like to thank Prof. L. elewski and Dr J. Popinigis for helpful suggestions.     

Photodynamic studies on acid ribonuclease from pea cotyledons

Summary In crude extracts, pea cotyledon acid ribonuclease is not inactivated by photodynamic treatment, but after 150-fold purification it is markedly inactivated when illuminated in the presence of rose bengal at pH 7.1. Data suggests that histidine photo-oxidation reduces catalytic activity.     

Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity

Summary Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA·DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.     

Labile protein-methyl ester: Comparison between chemically and enzymatically synthesized

Summary The rate of hydrolysis of protein-methyl ester, the enzymatic product of S-adenosylmethionine: proteincarboxyl methyltransferase (EC.2.1.1.24) acting on oxidized ribonuclease, was measured at pH 7.1 and 8.6 at 37°C. The half-life of the hydrolysis of the ester is 25 min at pH 7.1, and 4 min at 8.6. The rate of hydrolysis of the enzymatically formed esters at pH 7.0, in 0.1M phosphate buffer, was about 25 times faster than that of esters formed chemically by reaction with methanol in HCl. The lability of the enzymatically synthesized protein-methyl ester suggests that the esterification is specific to sites such that ionization of neighboring amino acid side chains enhances the rate of the hydrolysis.Acknowledgments. This work was supported by Research Grants AM 09603 from the National Institute of Arthritis and Metabolic Diseases, CA 10439 and CA 12226 from the National Cancer Institute, 1-P01-HD-05874 from the National Institute of Child Health and Human Development, National Institutes of Health, GM 20594-03 from National Institute of General Medical Sciences, USA.     

Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity.

Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA.DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

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.     

Class I and class II ribonuclease H activities inCrithidia fasciculata (protozoa)

Summary The protozoanCrithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg²⁺- and Mn²⁺- ions. However, the presence of Mn²⁺-ions is inhibitory for the Mg²⁺-dependent class II ribonuclease H activity ofCrithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.     

Detection and localization of a ribonuclease activity associated with vaccinia virus]

A ribonuclease associated with vaccinia virus can be detected when reduced concentrations of nucleotides are used for an in vitro RNA synthesis assay. The non-viral origin of this ribonuclease may be inferred from its external location and from its variable activity on different purified virus stocks. The detection of this ribonuclease activity on purified virus grown without foetal Calf serum may suggest that this enzyme is of cellular origin.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Class I and class II ribonuclease H activities in Crithidia fasciculata (Protozoa).

The protozoan Crithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg2(+)- and Mn2(+)-ions. However, the presence of Mn2(+)-ions is inhibitory for the Mg2(+)-dependent class II ribonuclease H activity of Crithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.     

Der Einfluss des Urin-pH auf die Aktivität von Urinenzymen

Summary The relationship between the pH of urine and the measured activity of ULDH, UAP and UAA was investigated in vivo and in vitro. The activity of ULDH and UAP is only half as much, or less, in acid urine (pH 4.8–5.1) as it is in alkaline urine (pH 7.0–8.1). The UAA activity, however, does not change in the pH range measured. The isoenzymes ULDH 2–4, present in alkaline urine of normal adults, does not appear in acid urine.     

Presence of a protein disulfide isomerase (EC 5.3.4.1) in wheat germ]

The presence of a protein disulfide isomerase (rearrangease) in wheat embryo has been demonstrated by its ability in reactivating randomly cross-linked ribonuclease. This activity requires a dialysable cofactor; after dialysis, the activity is recovered by addition of reduced glutathione. The enzyme can be precipitated by 70% saturation ammonium sulfate.     

Involvement of ribonuclease in the interactions of macrophages and fibroblasts in experimental silicosis

Summary Decreased ribonuclease activity in the supernatant from silica-treated macrophages is associated with the enhanced protein synthesis in granulation-tissue slices incubated in this supernatant, and with the decreased degradation of polysomes in granuloma slices and of polysomes isolated from the granulation tissue. The phagocytized silica particles adsorb ribonuclease and perhaps other proteins and thus remove them from the macrophage supernatant.For financial support we are grateful to the Association of Finnish Life Assurance Companies and to the Medical Research Council in Finland.     

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present. Received 26 October 2007; accepted 23 November 2007     

Esterase activity in the hepatopancreas ofMacrobrachium lamarrei (Crustacea: Decapoda)

Summary The activity of the hepatopancreatic esterase of the fresh water prawnMacrobrachium lamarrei was optimal at pH 7.4 and temperature 40°C. The activity increased with the increase in incubation period and enzyme concentration. The Michaelis constant (K_m) of the enzyme was 2.1×10^–3M.The investigations are a part of the thesis presented to University of Lucknow, India.Acknowledgments. The authors are grateful to the University Grants Commission, India for the award of a Junior Research Fellowship.     

Demonstration of fructose 1,6-bisphosphatase in human term placenta

Summary A proteolysed form of fructose 1,6-bisphosphatase (Fru-P₂ase) has been detected and characterized in human term placenta. The extract was found to contain very low levels of activity with an alkaline pH optimum. Western blotting demonstrated a polypeptide of Mr 26,000, instead of the subunit of Mr 36,000 observed in native mammalian Fru-P₂ases.     

Effect of metabolic versus respiratory acid-base changes on isolated coronary artery and saphenous vein

Summary Experiments were performed on helically cut strips from coronary artery and saphenous vein to determine the relative influence of metabolic versus respiratory acid-base changes. Tensions were measured over a range of various HCO₃ ^– concentrations and pCO₂'s. The results suggest that tension is influenced by extracellular pH and is independent of pCO₂.Supported by USPHS grant No. HL24232.     

The eosinophil ribonucleases

The eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3) are two closely related proteins with intriguing functional and evolutionary properties. While both EDN and ECP maintain the structural and catalytic residues typical of the RNase A superfamily, the role of ribonuclease activity in the physiologic function of these proteins remains unclear. The biochemistry and physiology of EDN, ECP and the recently discovered ribonuclease k6 (RNase 6) will be reviewed in this chapter.