The native molecular size of alkyl-dihydroxyacetonephosphate synthase and dihydroxyacetonephosphate acyltransferase.

Dihydroxyacetonephosphate acyltransferase (DHAP-acyltransferase) and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) are the first two enzymes involved in the biosynthesis of ether phospholipids. Both peroxisomal enzymes have recently been purified to homogeneity and their molecular weights under denaturing conditions were reported. To determine the in situ functional size of both enzymes, radiation inactivation experiments were performed. Alkyl-DHAP synthase showed single exponential decays, both when enzymatic activity and when immunoreactive protein levels were measured, from which target sizes of 79+/-2 kDa and 78+/-4 kDa, respectively, were calculated. DHAP-acyltransferase activity increased at lower doses and decayed upon further irradiation with an apparent target size of 62+/-7 kDa. We conclude from these data that the functional unit sizes for both enzymes in situ are represented by their single polypeptide chains.

Functional molecular mass of rat hepatic lipase in liver, adrenal gland and ovary is different.

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 degrees C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63 +/- 10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117 +/- 16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56 +/- 6 kDa and a target mass of 117 +/- 14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer.

Heat-stable enterotoxin receptor/guanylyl cyclase C is an oligomer consisting of functionally distinct subunits, which are non-covalently linked in the intestine.

Guanylyl cyclase (GC) C is a heat-stable enterotoxin (STa) receptor with a monomeric M(r) of approximately 140,000. We calculated from its hydrodynamic parameters that an active GC-C complex has a M(r) of 393,000, suggesting that GC-C is a trimer under native conditions. Both trimeric and dimeric GC-C complexes were detected by 125I-STa binding and SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The GC activity and STa binding from intestinal brush border membranes comigrated in gel filtration and velocity sedimentation with recombinant GC-C. However, 125I-STa cross-linking demonstrated that STa receptors with molecular masses of 52 and 74 kDa are non-covalently attached to GC in the intestine. Radiation inactivation revealed different functional sizes for basal GC activity, STa-stimulated GC activity, and STa binding (59, 210-240, and 32-52 kDa, respectively). At low radiation doses, basal GC activity was stimulated, suggesting that GC-C is inhibited by a relatively large, probably internal structure. These results suggest that STa may activate GC-C by promoting monomer-monomer interaction (internal "dimerization") within a homotrimeric GC-C complex, and that GC-C is proteolytically modified in the brush border membrane but retains its function.

A 30 kDa functional size for the erythrocyte water channel determined in situ by radiation inactivation.

The functional unit size of the water channel in rabbit erythrocytes was assessed using target size analysis following radiation inactivation. Using Radiochromic nylon dosimetry, accurate values of accumulated dose yielded an absolute target analysis, leading to direct determination of molecular size. The erythrocyte water channel functional size was shown to be 30 kDa, and is identical to the size found in rat renal proximal tubule brush border membranes (1), suggesting close homology of these two water channels. The result suggests that the 28 kDa channel-like intrinsic protein (CHIP28) recently isolated from human erythrocytes and proximal tubule (2), which is believed to form water channels of oligomeric construction may have a functional water channel activity in monomeric form.

Functional unit of 30 kDa for proximal tubule water channels as revealed by radiation inactivation.

The high water permeability of kidney proximal tubules is of paramount importance for isotonic reabsorption of 70% of the glomerular filtrate, and water channels have been postulated to account for the high water permeability. Target analysis following radiation inactivation was used to probe the molecular size of the water channel. Samples of brush border membranes from rat renal cortex were subjected to 3-MeV electron pulses from the Van de Graaff accelerator at a temperature of -130 degrees C. The inactivation of the renal brush border enzymes, alkaline phosphatase, and maltase was used for internal standardization of accumulated dose measurements in target analysis of the water channel. Osmotic water permeability was measured by following the change in scattered light intensity upon rapid mixing of vesicles with a hypertonic solution using stopped-flow spectrophotometry. The vesicle shrinkage response was biphasic and the rate of the fast phase decreased dose dependently by irradiation corresponding to a target size of 30 +/- 3.5 kDa. The total change in scattered light intensity was unaltered, indicating that irradiation did not destroy the lipid barrier. Our results provide strong support for the hypothesis that the high osmotic water permeability of renal proximal tubules results from a water channel-specific protein with a functional unit of 30 kDa.

Kinetics and mechanism of electron transfer between purines and pyrimidines, their dinucleotides and polynucleotides after reaction with hydrated electrons; a pulse radiolysis study.

The radical spectra of mixtures of thymidine 5'-monophosphate (TMP) or uridine 5'-monophosphate (UMP) with adenine 5'-monophosphate (AMP) after hydrated electron attack, measured from 5 to 3000 microsecond after pulse radiolysis, can only be described in terms of the radical spectra of the nucleotides if an electron transfer is taken into account from the purine radical anion to the pyrimidine, resulting in the formation of a pyrimidine radical anion. From analysis of the spectra of the dinucleoside phosphates ApU, dApT and dCpdA after eaq- attack it follows that the electron-donating species is the purine radical anion (A-.) rather than the protonated purine radical. The electron transfer competes with the fast protonation of the purine radical anion: A-. + py----A + py.- and A-. + H2O in equilibrium AH. respectively. The electron transfer is found to have a diffusion-controlled reaction rate constant of approximately 1.2 X 10(10) for TMP and 3.5 X 10(9) dm3 mol-1 s-1 for UMP.

Subnanosecond pulsing of a 3-MV Van de Graaff electron accelerator by means of a passive coaxial pulse shaper.

A passive coaxial pulse shaper has been developed which produces a subnanosecond duration pulse with short rise and decay time from a long pulse with short rise time. The mechanical construction of the pulse shaper is a modified coaxial air line T-section. The pulse shaper has been incorporated in the pulsing circuit of a 3-MV Van de Graaff accelerator. The form of the resulting electron beam pulses was monitored both as the charge collected by a coaxial target and as the Cerenkov light emitted by a quartz plate. In both cases sequential sampling techniques were used. The electron beam pulses were found to have rise and decay tiems of approxiamtely 100 ps and a half-width as short as approximately 200 ps could be obtained. An advantage of this method of producing subnanosecond beam pulses is that it does not interfere with normal nanosecond pulsed operation of the Van de Graaff.

On the radiation chemistry of some pyrimidine-electron adducts: reaction of pyrimidine negative ions with t-butanol radicals.

The rates of decay of the electron adducts of thymine, thymidylic acid and of uracil at pH approximately 8-5 and at wave-lengths of 340 or 350 nm in the presence of t-butanol as scavenger for OH radicals are not influenced significantly by the addition of 0-1 M NaClO4. It follows that the decay reactions do not take place between negatively-charged species. The best explanation seems to be an efficient reaction of the electron adducts with the t-butanol radicals formed by reaction of OH radicals with the t-butanol.