Purification and properties of rabbit liver phosphorylase phosphatase.

A procedure for the purification of rabbit liver phosphorylase phosphatase is described. The specific activity of the preparation is 2,100 units/mg of protein, representing a 25,000-fold purification. During the initial steps of the purification a large activation of enzyme activity was observed. The molecular weight of the purified enzyme was estimated by Sephadex G-75 chromatography to be 35,000, and by sucrose density ultracentrifugation to be 34,000 (2.9 S). On Na dodecyl-SO4 polyacrylamide disc gel electrophoresis a single component with a molecular weight of 34,000 was observed. The pH optimum is 6.9 to 7.4, and the Km for rabbit muscle phosphorylase alpha is 2 muM. The same procedure is also applicable to the extensive purification of phosphorylase phosphatase from rabbit muscle.     

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

EFFECT OF METHIONINE AND METHIONINE SULPHOXIMINE ON RAT BRAIN S-ADENOSYL METHIONINE LEVELS

Rat brain SAM levels were markedly increased after methionine administration, whereas the convulsant, L-methionine-dl-sulphoximine (MSO), produced a 35 per cent decrease in whole brain content of S-adenosyl-L-methionine (SAM). When methionine was given in combination with MSO, SAM levels were not decreased. Studies on the regional distribution of SAM revealed only a small variation between regions (from 24 nmol/g in midbrain to 49-5 nmol/g in striatum). SAM levels were reduced by about 50 per cent in the cerebellum, striatum, cortex and hippocampus 3 and 6 h after MSO. It is proposed that abberant cerebral methylation processes may be involved in the genesis of the MSO seizure.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

The role of malate dehydrogenase in adaptation to hypoxia in invertebrates]

In muscle tissue of lamellibranch molluscs and crustaceans (cf. Table for the species studied), high levels of malate dehydrogenase and low ones of lactade dehydrogenase were detected. There is a direct relationship between the value of MDH/LDH ratio and the capacity of organisms to withstand temporary anaerobiosis. Animals with high ratio may adapt to hypoxia by transition from aerobic metabolism to anaerobic one.     

Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions.

1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.     

Effect of aeration on lysine biosynthesis in nutrient media with different concentrations of components]

The lysine synthesis by the culture M. glutamicus T-3 on nutrient media with varying molasses concentrations was studied during cultivation under different aeration conditions. With an increase in the nutrient concentration the relationship between the lysine yield and aeration rate became very manifest. An elevation of aeration (Kv) from 1.2 to 6.3 g O2 1/hr increased the yield of lysine in the 15, 20 and 28% molasses medium by 3, 6 and 11 times, respectively. A decline in aeration decreased the biomass yield and increased the content of lactic acid and alanine in the culture liquid (to 19 and 4 g/l, respectively). The rate of respiration of the culture in the filtrate of the culture liquid measured in the Warburg apparatus depended on the cell age and molasses concentration in the nutrient medium and not on the aeration rate.