LG Ferren, RL Ward, and BJ Campbell. 1975. “Monoanion inhibition and 35Cl nuclear magnetic resonance studies of renal dipeptidase.” Biochemistry, 14, 24, Pp. 5280-5.Abstract
Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.
JK Martin, MG Luthra, MA Wells, RP Watts, and DJ Hanahan. 1975. “Phospholipase A2 as a probe of phospholipid distribution in erythrocyte membranes. Factors influencing the apparent specificity of the reaction.” Biochemistry, 14, 25, Pp. 5400-8.Abstract
The action of snake venom phospholipases A2 in intact human erythrocytes was investigated in detail. The basis phospholipase from Agkistrodon halys blomhifii was found to induce both hydrolysis of membrane phospholipids and total cell hemolysis under certain experimental conditions. The hydrolytic action of the basic enzyme was found to consist of two sequential events: (a) hydrolysis of 70% of the total cell ph osphatidylcholine without any evident hemolysis; and (b) complete hydrolysis of the remaining phosphatidylcholine, followed closely by extensive phosphatidylethanolamine hydrolysis and finally with onset of hemolysis, attack on the phosphatidylserine. At pH 7.4 and 10 mM Ca2+ only stage (a) occurred. However, a slight elevation of the pH of incubation to pH 8.0 and/or inclusion of 40 mM Ca2+ in the reaction mixture caused both events (a) and (b) to occur. The addition of glucose limited the action of the enzyme to stage (a) under any reaction conditions. An investigation showed that enzymically induced hemolysis occurred under conditions where the intracellular ATP levels were lowered. Data are presented which suggest that stage (b) is mediated by in influx of Ca2+ into the cell when the levels of ATP are low. Interestingly the phosphllipase from Naja naja venom (Pakistan) yielded results similar to those observed with the basic enzyme from Agkistrodon venom. However, the enzyme from Crotalus adamanteus and the acidic enzyme also present in the Agkistrodon venom produced only slight hydrolysis or hemolysis under any of the conditions studied. Other species of erythrocytes, e.g., guinea pig, monkey, pig, and rat, were tested but only those from guinea pig behaved similarly to the human cells. Pig, monkey, and rat erythrocytes underwent very limited hydrolysis and hemolysis. It is evident that the use of these phospholipases to probe the localization of phospholipds in erythrocyte membranes must be approached with caution. Certain facets of this problem are discussed.
RF Wachter, GP Briggs, and CE Pedersen. 1975. “Precipitation of phase I antigen of Coxiella burnetii by sodium sulfite.” Acta Virol, 19, 6, Pp. 500.
ES Elgart, T Gusovsky, and MD Rosenberg. 1975. “Preparation and characterization of an enzymatically active immobilized derivative of myosin.” Biochim Biophys Acta, 410, 1, Pp. 178-92.Abstract
Purified skeletal muscle myosin (EC has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.
W Siegenthaler, H Leutenegger, G Siegenethaler, and T Medici. 1975. “[Pulmonary involvement in collagen diseases].” Med Klin, 70, 45, Pp. 1801-14.
J Marniemi and MG Parkki. 1975. “Radiochemical assay of glutathione S-epoxide transferase and its enhancement by phenobarbital in rat liver in vivo.” Biochem Pharmacol, 24, 17, Pp. 1569-72.
B Hultberg, A Lundblad, PK Masson, and PA Ockerman. 1975. “Specificity studies on alpha-mannosidases using oligosaccharides from mannosidosis urine as substrates.” Biochim Biophys Acta, 410, 1, Pp. 156-63.Abstract
Oligosaccharides containing terminal non-reducing alpha(1 leads to 2)-, alpha(1 leads to 3)-, and alpha(1 leads to 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic alpha-mannosidases isolated from human and bovine liver. The enzymes released all the alpha-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than alpha-mannosidase B2, even though the apparent Km value for the substrates was the same with each enzyme. The human acidic alpha-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human alpha-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.
YW Chow, R Pietranico, and A Mukerji. 1975. “Studies of oxygen binding energy to hemoglobin molecule.” Biochem Biophys Res Commun, 66, 4, Pp. 1424-31.
R Saran, K Kishore, and R Viswanathan. 1975. “A study of parasites in domestic pests in households of patients with tropical pulmonary eosinophilia.” Indian J Chest Dis, 17, 3, Pp. 119-23.
ML Schwabbauer. 1975. “Use of the latent image technique to develop and evaluate problem-solving skills.” Am J Med Technol, 41, 12, Pp. 457-62.Abstract
This project involved designing, developing and evaluating a simulation module, utilizing the latent image technique. The general topic chosen for this simulation was the laboratory characterization of anemias. Target learner populations included medical technology students, physician assistant students, and pathology residents. Members of all three groups participated in the evaluation of the module and responded to its use in varied settings.