Decrease by cycloheximide of calcium binding and nonesterified fatty acids in rat-intestinal Golgi-enriched membrane fractions.

Rat intestinal Golgi-enriched membrane fractions bind more Ca2+ than do basolateral and microvillus-enriched membrane fractions, and this uptake is reduced by vitamin D-deficiency. The effect of the protein synthesis inhibitor, cycloheximide, on this Ca2+ binding was determined in rat fed a normal, vitamin D-sufficient diet. Cycloheximide, 1.5 mg/kg, rapidly reduced protein synthesis (measured by [3H]leucine incorporation) to 12% of control values within 15 min, but Ca2+ binding diminished gradually to 50% of control values by 60 min. Ca2+ transport across gut sacs was also decreased. The reduction in Ca2+ binding was not due to an alteration in vesicle morphology or to a direct effect of cycloheximide. Nonesterified (free) fatty acids, the probable binding sites for Ca2+ in these membrane fractions, were reduced by cycloheximide to 48% of control values by 60 min. There was no significant change in total lipid phosphate. Cycloheximide may affect the synthesis of proteins necessary for the presence of nonesterified fatty acids in these Golgi membranes.

Characterization of superoxide dismutase (SOD-1 and SOD-2) activities in inbred mice: evidence for quantitative variability and possible nonallelic SOD-1 polymorphism.

Liver Cu/Zn (SOD-1) and Mn (SOD-2) superoxide dismutase activities were determined in 12 inbred mouse lines. SOD-2 activity varied from 5 to 8 U/mg protein but was never more than 5% of the total. SOD-1 activity varied from 112 (SJL/J) to 155 (RF/J) U/mg protein, with the 12 strains falling into three activity classes. No correlation between SOD-1 activity and H-2 histocompatibility phenotype was observed, i.e., these two loci do not appear linked as previously suggested [Novak, R., Bosze, Z., Matkovics, B. and Fachet, J. (1980). Science 207:86]. Several tissues in all strains exhibited three SOD-1 charge electromorphs which did not differ in relative proportions between strains or tissues. The pI values of these three isozymes were 4.0, 4.5, and 5.0, respectively. The pI value of SOD-2 was 7.7. Both SOD-1 and SOD-2 were sensitive to CHCl3/EtOH extraction, but this sensitivity was not electromorph specific. Quantitation of the SOD-1 isozymic pattern indicated that the electromorphs were present at a ratio of 1:6:23 in order of increasing pI. Fitting of these data to a binomial distribution showed that they were consistent with the presence of two SOD-1 subunits (chains) of unequal pI. The mole fractions of the two chains were calculated to be 0.14 (lower-pI chain) and 0.86 (higher-pI chain). Since the mice used were highly inbred, this pattern could be due to unequal rates of transcription of linked, nonallelic SOD-1 loci, although other explanations are possible. The activity differences between SJL/J and RF/J appear large enough and the data precise enough to make genetic studies on the control of SOD-1 expression in the mouse practicable.

Kinetics of subcellular distribution in rat intestine of 1,25-dihydroxycholecalciferol administered in vivo. Evidence for concentration within 5 min into purified nuclei.

To better understand the initial steps in the induction of intestinal Ca2+ transport by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], we studied the early subcellular localization of 1,25(OH)2D3 in rat intestine. Vitamin D-deficient rats received 300 pmol of 1,25(OH)2[3H]D3 intravenously at 5 min to 4h before being killed. Cells homogenized in buffer of I = 90 mmol/litre were fractionated by centrifugation into a crude nuclear pellet, purified nuclei, Golgi and basal-lateral membranes, cytosol and a post-nuclear pellet. Nuclear purification was established by biochemical and morphological criteria and gave a yield of 32 +/- 2% (mean +/- S.E.M.; n = 21). Although re-establishment of Ca2+ uptake by Golgi is one of the earliest reported intestinal responses to 1,25(OH)2D3, no direct localization of 1,25(OH)2D3 to Golgi was detected. Purified nuclei had the highest specific radioactivity at all times studied, with nuclear localization detectable at 5 min and peak nuclear uptake at 1 h. Relative specific radioactivity of nuclei to cytosol increased from 5 min to 30 min, at which time equilibrium between cytosol and nucleus appeared to be attained. Nuclear uptake occurred in all cells from villus to crypt. Of total nuclear binding 10% was resistant to high ionic strength buffer (I = 365 mmol/litre); peak nuclear uptake was observed at 30 min in this buffer. This tight binding may represent the active fraction of 1,25(OH)2D3. These results indicate that localization of 1,25(OH)2D3 to rat intestinal nuclei precedes the observed Golgi-membrane effects and suggest the existence of high-affinity nuclear 1,25(OH)2D3-binding sites.

Genetic determination of amylase synthesis in the mouse.

Extensive genetic variation in structure and rate of synthesis of pancreatic amylase has been identified among strains of mice. The relative rate of synthesis of amylase varies from 0.15 in strain YBR to 0.26 in strain C3H. The number of electrophoretic isozymes of pancreatic amylase varies between one and four. In each strain with multiple amylase isozymes, a characteristic quantitative distribution of protein among the isozymes is observed. Isozyme proportions are determined by the relative rates of synthesis of each component. Congenic lines with different amylase phenotypes have been established. Genetic analysis reveals the close linkage of cis-acting sites determining rate of synthesis and electrophoretic mobility of mouse pancreatic amylase.

Additional evidence for the close linkage of amy-1 and amy-2 in the mouse.

The enzyme amylase is produced in large quantities in two mammalian tissues, the pancreas and the parotid salivary gland. A substantial body of biochemical and genetic evidence is consistent with the existence of distinct genes encoding salivary and pancreatic amylases, but testcrosses in mice have indicated that the two putative loci must be very closely linked. We have studied crosses between two pairs of inbred mice that differ with respect to electrophoretic mobility of salivary and pancreatic amylases. Among 343 potentially recombinant chromosomes examined, no recombinants were found. Our data sets an upper limit of 0.87 cM (P = 0.95) for the distance between the salivary and pancreatic loci.

Cadmium metabolism in cdm/cdm mice.

The uptake and metabolism of cadmium by cadmium-susceptible (+/+) and cadmium-resistant (cdm/cdm) strains of mice have been compared. These strains did not differ with respect to the quantitative uptake of cadmium into liver, kidney, or testis. After intraperitoneal administration of a nontoxic dose, more than 80% of the cadmium in liver and testis of both strains is bound to low molecular weight proteins. The chromatographic behavior of these cadmium-binding proteins is not affected by cdm genotype.