A simple,rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation

The Multiskan spectrophotometric system and Coomassie brilliant blue G-250 protein-dye binding method have been used together to measure NaOH-solubilized protein in subcellular membrane fractions prepared from isolated rat adipose cells. Forty-eight samples can be read in duplicate within 1 min. Sucrose in concentrations up to 0.7 m interfere only moderately with the assay. A rapid and convenient method is, therefore, now available for multiple protein determinations following sucellular fractionation on sucrose gradients.     

The cellular dynamics of hepatic receptors for alpha 2-macroglobulin.protease complex and for insulin are different

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.     

Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Stimulatory role for endogenous opioid peptides on postexercise insulin secretion in rats

Endogenous opioid peptides (EOP) and prior exercise may modulate the stimulatory effect of glucose on insulin secretion. To gain insights into these relationships, we studied male Wistar rats (187-245 g) during sustained hyperglycemia by use of the glucose clamp technique. Four groups of sedentary fed rats (n = 8/group) either ran (Ex) at 24 m/min, 0% grade, or rested (R) for 40 min. Thirty minutes after Ex or R, arterial blood glucose was elevated to and maintained at 11 mM for 2 h by a variable glucose infusion. At the start of Ex or R rats had saline (Sal) or naloxone (Nal, an opioid antagonist) intravenous infusions for 160 min (40 min Ex + 30 min R + 90 min of a 120-min glucose clamp). Steady-state glucose infusion rates (SSGIR) were approximately 55 mg.kg-1.min-1 at the start of the clamp and declined significantly over the 2nd h to approximately 45 mg.kg-1.min-1. No significant differences existed in SSGIR between groups. R-Sal and Ex-Sal groups did not differ in their insulin response to hyperglycemia. In contrast, when all groups were compared at the end of the Nal or Sal infusion, Ex-Nal had the lowest insulin concentration (749 +/- 174 pmol/l), whereas the R-Nal group had the highest (1,581 +/- 216 pmol/l, P less than 0.05). These data suggest a stimulatory role for EOP on insulin secretion that is expressed after a prior stress (Ex). Thus one function of exercise-induced activation of EOP may be to regulate insulin secretion in the immediate postexercise period.     

Comparison of the Action of Ampicillin and Benzylpenicillin on Enterococci In Vitro

The bactericidal activity of benzylpenicillin and ampicillin on 21 strains of enterococci was evaluated and compared to the activity of these drugs in combination with streptomycin (20 mug/ml). On a weight basis, ampicillin was about twice as effective as benzylpenicillin. Neither of the drugs was rapidly and completely bactericidal for any of the 21 strains of enterococci when used alone. The addition of streptomycin greatly enhanced the early bactericidal rate achieved with any given amount of either penicillin and permitted the elimination of viable organisms in vitro. These results suggest that, for the time being, combined antibiotic therapy might be desirable in enterococcus endocarditis and that ampicillin, although more effective than benzylpenicillin, should not be relied upon as a single drug in that disease.     

Internalization of insulin and its receptor in the isolated rat adipose cell. Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 degrees C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [125I]insulin to these same membrane fractions. At 37 degrees C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20-30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16 degrees C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.