Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Origin of mitochondrial enzymes. V. The polypeptide character and the biosynthesis of rat liver cytochromec oxidase polypeptides by mitochondria

Isolated rat liver mitochondria were labeled in vitro withl-[¹⁴C]leucine. Sixty percent of the incorporated radioactivity was found to reside in subunits 1, 2, and 3 of cytochromec oxidase with apparent molecular weights of approximately 33,000, 25,000, and 20,000, respectively. The results indicate that these are the predominant products of protein synthesis under the conditions employed. The enzyme complex, as derived by immunoprecipitation, was found to contain four additional polypeptides with apparent molecular weights of 17,000, 12,500, 7000, and 3500. A comparison of electrophoretic profiles of the rat liver and beef heart enzyme reveals that the apparent molecular weights of all polypeptides are remarkably similar.To be submitted as partial fulfillment of the requirements for the Ph.D. degree of this institution.     

Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict “flipping” across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity. The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide- or nitroxide-bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.     

Effect of vasotocin on plasma free fatty acid level in the migrating anadromous sea lamprey.

The effect of injections of arginine vasotocin (AVT) on plasma free fatty acid (FFA) levels was studied in anadromous sea lampreys collected in the St. John River, New Brunswick, during their upstream spawning migration. Plasma FFA was significantly higher in lampreys injected with a single dose of 1 000 mU vasotocin/kg body weight than in those receiving only the vehicle solution, the difference being the greater at 90 than at 30 min post-injection. The significance of AVT in migration is discussed.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: Investigations into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to exogenously supplied fatty acids

The regulation of the nature and quantity of the fatty acids produced in vivo by Acholeplasma laidlawii B in the presence of various exogenous fatty acids has been investigated. In the presence of exogenous medium- or long-chain fatty acids, the organism appears to reduce the amounts of de novo biosynthesized fatty acids in its cellular lipid pool by two distinct mechanisms: an excretion of biosynthesized fatty acids to the growth medium as free fatty acids, and a reduction in total de novo biosynthetic output. These two mechanisms do not suffice to maintain constant total membrane lipid levels, but they do appear to significantly moderate the effect of exogenous fatty acids on the level of membrane lipid. In the presence of short-chain fatty acids, total membrane lipid levels are not elevated. Exogenous fatty acids can cause shifts in the average chain length of de novo biosynthesized fatty acids; the magnitudes and directions of these shifts can be correlated with the specificity of the exogenous species for esterification to the 1- or the 2-position of the glycerol moiety of membrane glycerolipids. As the various endogenously synthesized fatty acids differ in their positional specificity for glycerolipid esterification, we propose that the competition of an exogenous species with significant specificity for a particular position with the endogenously derived fatty acids specific for that position can selectively depress the synthesis of such endogenously derived species, thereby altering the overall product spectrum of de novo fatty acid biosynthesis in vivo.     

Pressure jump relaxation kinetics of frog skin open circuit voltage and short circuit current

The relaxation kinetics of frog skin open circuit voltage, V_oc, and short circuit current I_sc, was studied by analyzing the effects of subjecting the tissue to sudden increments of hydrostatic pressure. Both V_oc and I_sc are perturbed by the pressure jump. Changes in V_oc can be resolved into three components: a rapid decrease (phase I), a second, additional decrease with time constant 2.2 s (phase II), and finally a very slow increase found only in some preparations. The amplitudes of phases I and II are linear in the range of pressures studied (<350 atm) and have respective pressure coefficients of −1.2 · 10⁻⁴atm⁻¹ and −3.7 · 10⁻⁴atm⁻¹.Under short circuit conditions phases I and II persist. The pressure coefficients of the amplitudes of phase I and II, −4.3 · 10⁻⁴atm⁻¹ and −5.0 · 10⁻⁴atm⁻¹, respectively, are larger than those of V_oc, but the time constant of phase II, 2.2 s, is the same. The sum of the amplitudes of phases I and II is directly proportional to I_sc when it is inhibited with ouabain. It is argued that in both electrical states pressure perturbs the same transport mechanism giving rise to phases I and II of V_oc and I_sc.The magnitude of the pressure coefficients of these processes implies that they arise from chemical reactions, rather than from simple, physical solution properties. Comparison of the pressure jump kinetics with the previous spectral analysis of the electrical fluctuations of frog skin suggests a common origin for both sets of phenomena.