Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Host-parasite Interrelationship in Phosphatase Activity

Infection by Cuscuta and Orobanche causes significant losses in dry solids and protein content in host plant or plant part. Changes occur in phosphatase activity towards fructose-1,6-diphosphate at alkaline pH and β-glycerophosphate at acid pH, expressed per mg protein or g fresh tissue. The leaves of all hosts infected by Orobanche show an increase in the alkaline fructosediphosphatase activity, whereas as far as the infection by Cuscuta is concerned the general response is a decrease in the enzyme in the shoots. The alterations in the phosphatase activity towards β-glycerophosphate at acid pH in the shoots are not consistent. However, there is a marked increase in the acid phosphatase activity against β-glycerophosphatase in the roots of the infected hosts. The significance of these findings has been discussed in the light of host-parasite interrelationship.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

A causal approach to the study of fertility and familism

This paper tests, within the framework of LISREL, the causal structures of fertility using data from the 1973-74 Growth of Alberta Family Study (GAFS) of women aged 18-44 who are currently married or living common-law. Differential fertility among two groups of women classified by nativity also are examined. The women's background characteristics (e.g., age, religiosity, and education) are viewed as exogenous variables. The endogenous variables are familism and expected family size; familism is designated as an intermediate variable in the model, linking demographic and socioeconomic (including cultural) factors to fertility, The results indicate that familism acts as an important variable explaining fertility, particularly, among foreign-born women. The study confirms and extends earlier research findings that religiosity and education influence couples' fertility, the former positively and the latter negatively.     

Recent developments in in vivo neutron activation analysis facilities

Two new facilities for in vivo activation analysis of patients have been designed, developed, and constructed at Toronto General Hospital. One of these is for the determination of body calcium for the diagnosis of osteoporosis and other diseases associated with bone loss. The other is for the measurement of total body nitrogen for the determination of protein status. These facilities replace old university facilities and take into account the comfort and management of patients. In addition, in the case of the calcium facility, the precision of the measurements has been improved because of larger detector volume and increased neutron source strength. Both the facilities are now in routine hospital clinical use.     

Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1'' binding site.

Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged at R values of 0.158 in the 7.0-2.3-A range for RWJ-50353 and 0.155 in the 7.0-1.8-A range for RWJ-50215. Interactions between the protein and the thiazole rings of the two inhibitors provide new valuable information about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-binding benzothiazole group linked to the D-Phe-Pro-Arg chloromethyl ketone motif. Interactions with the S1-S3 sites are similar to the D-phenylalanyl-prolyl-arginyl chloromethylketone structure. In RWJ-50215, a S1'-binding 2-ketothiazole group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity surrounded by His57, Tyr60A, Trp60D, and Lys60F. This location of the S1' binding site is consistent with previous structures of thrombin complexes with hirulog-3, CVS-995, and hirutonin-2 and -6. The ring nitrogen of the RWJ-50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen bond with the NZ atom of Lys60F.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue α-amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 α-carbon in the d conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue -amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 -carbon in the conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.