Bovine seminal ribonuclease: Non-hyperbolic kinetics in the second reaction step

Peroxisomes contain enzymes catalyzing the β-oxidation of fatty acids, which have been purified and partially characterized. Hypolipidemic drugs, including clofibrate, cause a marked proliferation of peroxisomes and a striking increase in the activity of their β-oxidation system. We have compared by sodium dodecyl sulfate—polyacrylamide gel electrophoresis the polypeptide patterns of normal and clofibrate-induced peroxisomes and the purified β-oxidation enzymes. The data allow a tentative identification of the β-oxidation enzymes among the peroxisomal polypeptides; these enzymes constitute only a small part of the protein of normal peroxisomes. A subset of peroxisomal polypeptides, including the β-oxidation enzymes, is preferentially increased by clofibrate.     

The first fifty years of the Connecticut Tumor Registry: reminiscences and prospects

The first fifty years of the Connecticut Tumor Registry (1935-1985) have seen unprecedented progress in the collection of standardized data on cancer patients and in the processing of these data, from paper documents to punch cards and magnetic tapes. The need for collecting such information was first recognized, in the early 1930s, by a group of physicians, health professionals, and laymen in New Haven who observed alarming increases in cancer rates and poor survival of cancer patients in this city. This paper recalls the growth and development of the registry and the role played by the Connecticut legislature, the State Medical Society, the Connecticut Department of Health, and the National Cancer Institute in this process. For half a century, the registry has provided assistance to practitioners, hospitals, and research scientists, not only in Connecticut but across the country and around the world. By making available reliable data on incidence and survival, the registry has played a key role in patient management, clinical trials, and etiologic studies. It has also demonstrated the value and served as an exemplary model of a population-based registry. At this juncture in its history, prospects for the future of the Connecticut Tumor Registry appear bright. Its data base will be an essential resource for the recently established Cancer Control Research Unit (CCRU) in the state and for new intervention studies by investigators at Yale, the University of Connecticut, and the State Health Department.     

Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.     

British mammal populations: fifty years of change

• 1 Agricultural intensification, urbanization, overpopulation by both native and invasive species and diseases have all had marked effects on populations of British mammals over the last 50 years.
• 2 Here, accounts are presented of the effects of these processes with a prospective view of what the future holds for the mammals of Britain.

     

Conformation properties and enzymatic activity of pancreatic ribonuclease in soluble complexes with sodium dextran sulfate

Effect of complex formation with dextran sulfate (DS) (substitution degree 1.3, molecular mass 500 thousand) on RNAse enzymic activity. its spatial structure and conformation stability was studied. Hydrolytic activity of the enzyme in complex in inhibited already at small additions of DS, while the transferase one is changed only at a great excess of the polyelectrolyte. It has been shown by CD spectra that no notable conformation changes proceed in the enzyme during complex formation, although the enzyme turns destabilized to the denaturing effect of heat at the expense of strengthened interactions between DS and RNAse during its denaturation. Thus the inhibition of hydrolytic activity in the complex is primarily related to limitations for the formation of the enzyme-substrate complex on polyelectrolyte charged likely with the substrate, and not to the protein conformation changes.     

Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors.

The aim of this study was to evaluate pancreatic juice secretion of calves in the first postnatal days, and determine a potential involvement of cholecystokinin (CCK) and intestinal CCK receptor in its regulation. Nine neonatal Friesian calves (five controls and four treated intraduodenally with FK480, a CCK-A receptor antagonist) were surgically fitted with a pancreatic duct catheter and a duodenal cannula before the first colostrum feeding. Collections of pancreatic juice and duodenal luminal pressure recordings were started early after recovery from anaesthesia and continued for 6 days. From day 2 or 3 of life, periodic fluctuations in pancreatic secretions were observed in concert with duodenal myoelectric motor complex (MMC) and variations in plasma pancreatic polypeptide (PP) concentrations. Intraduodenal administration of FK480 reduced pancreatic juice secretion while intravenous infusion of CCK had no effect. Immunocytochemistry indicated an association of mucosal CCK-A and -B receptors with neural components of the small intestine. In conclusion, periodic activity of the exocrine pancreas exists in neonatal calves soon after birth and local neural intestinal CCK-A receptors could be partly responsible for the modulation of neonatal calf pancreatic secretion.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.     

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 mouse pancreatic ribonuclease

Mouse pancreatic ribonuclease has been crystallized in a form suitable for X-ray structure determination. The crystals grown from solutions of 2-methyl-2,4-pentanediol diffract to high resolution and belong to the hexagonal space group P6(1) (P6(5)) with unit cells dimensions a = b = 64.44 A, c = 53.91 A, y = 120 degrees and V = 1.94 x 10(5) A3 (1 A = 0.1 nm). There are six molecules per unit cell (1 molecule/asymmetric unit), and Vm = 2.3 A3/dalton.     

The kinetics of pancreatic ribonuclease reaction with alkaline and acidic forms of poly A

Summary The RNase hydrolysis of random-coil (alkaline form) poly A follows biphasic kinetics at low salt concentrations. However, its resistance to RNase increases with the ionic strength. Helical (acidic form) poly A is also susceptible to RNase but its hydrolysis follows first-order kinetics, and its resistance increases as the pH is lowered. These conformation-dependent kinetics of poly A hydrolysis are similar to those obtained in the hydrolysis of cellular RNA and reovirus double-stranded RNA.