Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.     

Phosphodiesterase in Microsomes from Bovine Milk

Phosphodiesterase was solubilized from bovine milk microsomes and partially purified. The purified enzyme showed 20-fold specific activity compared with that of microsomes, and 1,500-fold with that of the original milk.

The properties of the enzyme were investigated by using NpT. The pH optimum was at 9.5. The enzyme was inhibited with EDT A and reactivated with the addition of magnesium or calcium ions. This enzyme was strongly inhibited with reducing reagents. K_m, value was 7.4 x 10^-4 M for NpT at pH 9.5.

RNA was hydrolyzed completely to 5′-mononucleotides, and this enzyme may be considered to show the exonucleolytic action for RNA.     

Acid Protease of Bovine Milk

Occurrence of milk acid protease in bovine casein in addition to alkaline protease was found and purification of this enzyme was achieved. The enzyme had a pH optimum at 4.0 and was most stable at pH 3.5. The molecular weight of the enzyme was 36,000 and no inhibition was observed by diisopropyl-fluorophosphate, EDTA etc. This enzyme is considered to be similar to cathepsin D.

Milk acid protease mainly hydrolyzed α_s-casein and similar change was observed in autolysis of casein at pH 5.5. It is suggested that milk acid protease may have some significance in cheese ripening.     

Occurrence of Superoxide Dismutase in Bovine Milk

Acidic heteropolysaccharides, d-glucurono-d-xylo-d-mannans were isolated from the water- and alkaline extracts of the fruit body of Tremella fuciformis Berk. Similar polysaccharides were isolated from the growing culture of the haploid cells of two strains (T–19 and T–7) of T. fuciformis, when they were cultured in sucrose or glucose-yeast extract medium. The extracellular polysaccharides contain, d-glucuronic acid, d-xylose and d-mannose [molar ratios, 1.3: 1.0: 3.5 (T–7) and 0.8: 1.0: 2.1 (T–19)], and, in addition, small proportions of l-fucose and O-acetyl groups. Methylation and Smith degradation studies indicated that both fruit body and extracellular polysaccharides are built up of α-(1 → 3)-linked d-mannan backbone chain to which β-linked d-glucuronic acid and single or short chains of β-(1 → 2)-linked d-xylose residues are attached at the C–2 position. l-fucose residues in the extracellular polysaccharides may form the single branches. The structural features of these polysaccharides are discussed in comparison with the similar polysaccharides from other fungi.     

High Molecular Weight Mucin-like Glycoprotein in Bovine Milk

We purified an enzyme hydrolyzing 2-sulfo-α-L-fucopyranose pyridylaminated 2-sulfo-α-L-fucopyranosyl-(1→2)-fucose from the acetone powder of the digestive tract of a sea urchin. The enzyme was purified 307-fold with an overall recovery of 1.63% by ammonium sulfate precipitation, cation exchange chromatography, and gel filtration chromatography. The enzyme is useful for the structural analysis of sulfated fucan.     

Comparison of Bovine Milk Protease with Plasmin

Comparative studies of bovine milk protease and bovine plasmin were performed. It was found that milk protease was very similar to plasmin in various properties such as optimum pH, pH-stability, heat-stability, inhibition by various inhibitors and molecular weight. The changes of casein by both enzymes as observed by polyacrylamide gel electrophoresis were also quite similar. From these results, it is suggested that milk protease may be plasmin itself transported from bovine plasma.     

The Electrical Conductivity of Bovine Milk in Mastitis Diagnosis

The electrical conductivity (EC) of milk is mainly a function of the electrolyte concentration in the milk and therefore raised in mastitis. The present investigation was aimed at elaborating, if possible, a diagnostic model for screening purposes based on EC determinations and consistent with the diagnostic procedures and interpretations commonly used in laboratory milk diagnosis in the Nordic countries (Klastrup 1975). According to this diagnosis (here called reference diagnosis) cell numbers above 300,000/ml (cell count or the corresponding CMT-score) in foremilk quarter samples during the main part of the lactation period and significantly above the lowest value on within-udder comparison during late lactation are considered indicative of mastitis and bacteriological examinations are made when called for.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

精氨酸对牛胰核糖核酸酶氧化复性的时相性抑制作用

精氨酸常在重组蛋白的体外复性中,作为一种小分子添加剂用于抑制蛋白聚集。精氨酸对蛋白复性折叠过程本身的影响尚不清楚。首次以不易聚集的牛胰核糖核酸酶为研究对象,通过飞行质谱检测氧化复性中间体的变化,通过酶学活性检测蛋白活性的恢复过程,观察了精氨酸对氧化复性的直接影响。发现不同浓度精氨酸对牛胰核糖核酸酶的氧化复性有直接的抑制作用。特别在0.5 mol/L浓度时,精氨酸对牛胰核糖核酸酶氧化复性的抑制作用具有独特的时相依赖性:早期复性可持续至4 h,其活性恢复达30%后,晚期复性基本终止。这种抑制特征与脲的抑制作用有明显的不同,精氨酸没有完全抑制关键结构中间体的形成。结果说明,牛胰核糖核酸酶的氧化复性,除经关键结构中间体的主要通路外,还可能存在一个潜在复性通路,它是其氧化复性后期的关键限速通路。该复性通路可以被0.5 mol/L精氨酸完全阻断。     

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

The Chemical Structure of Glycolipids of Bovine Milk

Experiments were executed to elucidate the chemical structure of ceramide monohexoside (CMH) and ceramide dihexoside (CDH) isolated from cow’s milk, especially with regard to the nature of the sugar moiety of the molecules. The results have shown that the structure of CMH and CDH in bovine milk is β-glucosyl-(l→l)-N-acyl-sphingosine, namely ceramide glucoside, and β-galactosyl-(1→4)-β-glucosyl-(1→1)-N-acyl-sphingosine, namely ceramide lactoside, respectively.     

Reduction of Nucleic Acid Content in Candida Yeast Cells by Bovine Pancreatic Ribonuclease A Treatment

Yeast as a source of protein for human consumption is limited by its relatively high nucleic acid content. In this study, we developed an enzymatic method of decreasing the nucleic acid content. Candida utilis cells, heat-shocked at 80 C for 30 sec, were treated with bovine pancreatic ribonuclease A. Maximum leakage of nucleic acid was observed when the incubation temperature was between 55 and 65 C, the pH of the system from 6.75 to 8.0, and the enzyme-to-cell ratio 1:10,000 on a weight-by-weight basis. Other factors, such as yeast strain, age of cells, and method of propagation, did not influence the susceptibility of the yeast cells to the action of ribonuclease. Buffers and monovalent cations had no inhibiting effects. Magnesium and calcium ions at concentrations greater than 0.001 m showed marked inhibition on the rate of nucleic acid leakage. This enzymatic method reduced the nucleic acid content of yeast cells from 7.5 to 9.0% to 1.5 to 2.0% with no significant concomitant loss of protein.     

Milk Antitrypsin Activity During Clinical and Experimental Bovine Mastitis

Milk trypsin-inhibitor levels were followed during clinical and experimental endotoxin-induced mastitis. Milk trypsin-inhibitor level proved to be a good indicator of mastitis, closely relating to milk BSA values. The trypsin-inhibitor pattern in the quarter milk samples during mastitis closely resembled the pattern of BSA, which indicates that the trypsin-inhibitor is blood derived and diffuses to the mammary gland lite albumin. The timing of the increase in the trypsin-inhibitor into the endotoxin infused quarter closely matched with the increase in body temperature, heart rate, decrease in blood leucocytes and increase in somatic cells. Serum zinc and iron showed a decrease a few hours later.     

Isolation and Biological Properties of Osteopontin from Bovine Milk

A procedure for the isolation of osteopontin (OPN) from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded ∼8 mg of purified protein per liter of milk. SDS–PAGE analysis revealed that the protein migrated atM_r60,000. NH₂-terminal sequence analysis of the first seven amino acids revealed the protein to be identical to that previously reported for bovine OPN. Also, our preparation demonstrated expected biological properties of OPN including adhesion of both endothelial and vascular smooth muscle cells to OPN in a dose- and Arg-Gly-Asp-dependent manner. Furthermore, OPN coupled to Sepharose was capable of binding the α_vβ₃integrin from a detergent extract of endothelial cells. Thus, our procedure yielded biologically active OPN from an abundant and natural source.     

Role of Ribonuclease and Ribonuclease Inhibitor Activities in Alzheimer''s Disease

It has been suggested that defects in the relationship between ribonuclease and its proteinaceous inhibitor could be a contributory factor in Alzheimer's disease. We have investigated this possibility further by analysing free and bound enzyme activities and the activity of the inhibitor in nine regions of diseased and normal brain. These were chosen to include areas known to be affected by the disease, regions not histologically affected but thought to be involved in the disease process, and areas not thought to be involved in the disease. Neither the enzyme nor its inhibitor is defective in its activities in the chosen areas of Alzheimer's disease brain when compared with those of carefully age-matched controls.     

牛乳碱性蛋白二维凝胶电泳条件的优化

为了在二维凝胶电泳(2-DE)中有效分离牛乳中的碱性蛋白,本试验在等电聚焦上样缓冲液中加入异丙醇和甘油,采用杯上样方式,比较了三丁基磷(TBP)和二硫苏糖醇(DTT)两种还原剂对碱性区域蛋白的分离效果。结果发现,等电聚焦上样缓冲液以TBP为还原剂的碱性蛋白分离图谱水平条纹少,优于以DTT为还原剂图谱。表明等电聚焦上样缓冲液以TBP为还原剂,采用杯上样的方法可以改善牛乳中碱性蛋白的分离效果。     

Ribonuclease inhibitors

RNases are important enzymes of cell metabolism, influencing gene expression, affecting cell growth and differentiation, and participating in cell defense against pathogens and induction of apoptosis. Since RNases mostly occur in complex with their inhibitors in the cell, the inhibitors also play a role in the above processes. The review considers natural protein RNase inhibitors of animals, plants, and bacteria, as well as synthetic low-molecular-weight inhibitors. Special emphasis is placed on the prospective use of RNase inhibitors in the therapy of cancer and allergy. While RNases are widespread, the number of the available natural and synthetic inhibitors is limited. A pressing problem is to design highly effective low-molecular-weight inhibitors of the RNase activity of angiogenin and eosinophil-associated RNases for anticancer and antiallergy therapy.