In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities.

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.

Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system.

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S-adenosylhomocysteine hydrolase and 5'-methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non-thermal microwave effects. The exposure causes a non-thermal, irreversible and time-dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S-adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

Functional lysosomal hydrolase size as determined by radiation inactivation analysis.

Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.

[The effect of exposure to 60Co accelerated electrons and gamma quanta on the activity of oxidative and hydrolytic enzymes in the rat brain]. / Vliianie vozdeistviia uskorennykh élektronov i gamma-kvantov 60Co na aktivnost' okislitel'nykh i gidroliticheskikh fermentov golovnogo mozga krys.

In experiments with 112 male Wistar rats it was shown that accelerated electrons (85 Gy) caused a significant increase in activities of succinate dehydrogenase (SDG) by 15.8% and lactate dehydrogenase (LDG) by 17.0%, and a decrease in activities of alkaline phosphatase (AP) and monoamine oxidase (MAO) by 10.6 and 7.8% respectively within the sensorimotor region of the cerebral cortex immediately after irradiation. Activity of SDG and MAO decreased (by 16.4% and 7.8% respectively) in the caudate nucleus over the same period of time. An increase in the accelerated electron dose from 85 to 500 Gy did not change the direction and the rate of the radiation response of the enzymes. Exposure of rats to 60Co gamma quanta (75 Gy) increased SDG and LDG activity (by 21.4 and 17.3% respectively) within the sensorimotor cortex as late as 10 min after irradiation. A repeated significant increase in SDG and LDG activity was observed 2 hr after irradiation.

Photoaffinity labeling of human placental S-adenosylhomocysteine hydrolase with [2-3H]8-azido-adenosine.

The potential photoaffinity probe 8-azido-adenosine (8-N3-Ado) was shown to serve as a substrate for the 3'-oxidative activity of human S-adenosylhomocysteine (AdoHcy) hydrolase (Aiyar, V. N., and Hershfield, M. S. (1985) Biochem. J. 232, 643-650). In this study, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified. After irradiation of the reaction mixture of [2-3H]8-N3-Ado and AdoHcy hydrolase (NAD+ form) and followed by tryptic digestion, peptides specifically photolabeled by [2-3H]3'-keto-8-N3-Ado were effectively separated from peptides nonspecifically labeled with [2-3H]8-N3-Ado using boronate affinity chromatography. After purification by reverse phase high performance liquid chromatography, two photolabeled peptides were isolated and identified as Val175-Lys186 and Val319-Arg327, in which Ala177 and Ile321 were associated with radioactivity. The specificity of the photoaffinity labeling with [2-3H]3'-keto-8-N3-Ado was demonstrated by the observation that these photolabeled peptides were not isolated when [2-3H]8-N3-Ado was incubated with apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are assumed to be parts of the adenine-binding domain for substrates. They are both within well conserved regions of AdoHcy hydrolases. The peptide Val175-Lys186 is located very close to Cys195 and Glu197. Ser198, both of which were indicated to be located in the active site of the enzyme by chemical modification and limited proteolysis methods. The peptide Val319-Arg327 is adjacent to Leu330, which is proposed by a computer graphics model to interact with the C-6-NH2 group of Ado.

[Changes in the activity of the oxidative and hydrolytic enzymes of the animal cerebral cortex in the early periods after gamma irradiation and the use of meksamine and the synthetic analog of prostaglandin F2 alpha-estrofan]. / Izmenenie aktivnosti okislitel'nykh i gidroliticheskikh fermentov korygolovnogo mozga zhivotnykh v rannie sroki posle gamma-oblucheniia v usloviiakh primeneniia meksamina i sinteticheskogo analoga prostaglandina F2 al'fa-éstrofana.

In experiments with (CBA x C57B1/6)F1 mice it was shown that LDH activity moderately increased 5 min after exposure of the head to 200 Gy gamma radiation. After 60 min, there was a 24.4 per cent decrease in alkaline phosphatase activity and a 24.3 per cent increase in SDG activity. Injected prior to irradiation meksamine precluded the postirradiation increase in SDH and alleviated the postirradiation decrease in alkaline phosphatase.