Frances M. Finn

Oxytocin and Vasopressin are Present in Human and Rat Pancreas

Immunoreactive oxytocin and vasopressin were found in human and rat pancreatic extracts. The pancreatic oxytocin and vasopressin eluted on Sephadex G-75 gel filtration chromatography and on reverse phase high pressure liquid chromatography in the same positions as their respective reference preparations. The immunoreactive oxytocin was biologically active in the rat milk ejection assay. The presence of oxytocin and vasopressin in human and rat pancreatic extracts suggests the possibility of local synthesis of both hormones. The neurohypophysial hormones are known to be endocrine mediators of insulin and glucagon release. The finding of oxytocin and vasopressin in the pancreas raises the possibility, although yet unproven, of local synthesis and perhaps a paracrine function for the neurohypophysial peptides upon pancreatic hormone release or for a local function upon the liver.

Vitamin K uptake in hepatocytes and hepatoma cells

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.

Studies with the S-peptide-S-protein system: The role of glutamic acid-2, lysine-7, and methionine-13 in S-peptide1–14 for binding to and activation of S-protein☆☆☆★

Abstract Syntheses are described of 12-serine S-peptide 1–14 , 12-(β-pyrazolyl-3)-alanine S-peptide 2–14 , 12-(β-pyrazolyl-3)-alanine S-peptide 3–14 , 13-norleucine S-peptide 1–14 , 13-norleucine S-peptide 2–14 , 13-norleucine S-peptide 3–14 , 7-norleucine S-peptide 1–14 , and 7,13-dinorleucine S-peptide 1–14 . The peptides were prepared by combination of the stepwise method and fragment condensation and were characterized by thin-layer chromatography and amino acid analyses of acid and enzymic hydrolysates. The interaction between these peptides and S-protein or ribonuclease S was determined with RNA as the substrate. The peptides in which 12-histidine is replaced by serine or β-(pyrazoly--3)-alanine did not activate S-protein but had the ability to compete with S-peptide 1–20 for S-protein and thus inhibited RNase S. 12-(β-Pyrazolyl-3)-alanine S-peptide 2–14 was essentially as effective an inhibitor of RNase S as 12-(β-pyrazolyl-3)-alanine S-peptide 2–14 ; 12-(β-pyrazolyl-3)-alanine S-peptide 3–14 was about 30-fold less active. 13-Norleucine S-peptide 1–14 and 13-norleucine S-peptide 2–14 activated S-protein and were considerably more effective than 13-norleucine S-peptide 3–14 . Together with previous observations, these results firmly establish the “binding” contribution of glutamic acid-2 in the S-peptide-S-protein system. A possible explanation for this phenomenon is presented. 12-Serine S-peptide 1–14 was approximately sevenfold less effective an inhibitor as 12-(β-pyrazolyl-3)-alanine S-peptide 1–14 . This finding indicates that the aromatic azole ring of histidine and of β-(pyrazolyl-3)-alanine contributes to the stability of the respective S-peptide-S-protein complexes. The observation that both 7-norleucine S-peptide 1–14 and 7,13-dinorleucine S-peptide 1–14 activate S-protein does not support the suggestion that lysine-7 is an important substrate binding site in RNase S.

Isolation and characterization of hormone receptors.

Publisher Summary Remarkable progress in the techniques of molecular biology has advanced the field of protein sequencing immeasurably. A highly specific isolation method, such as affinity chromatography is essential to the success of such an undertaking. The nonspecific attachment of peptide hormones to activated Sepharose resins can generate a number of different species with varying affinities for the desired receptor. There are a number of advantages to the avidin–biotin technique. The formation of affinity column is highly specific—namely, through the avidin–biotin interaction and is achieved by simply mixing the biotinylated ligand with the Sepharose-immobilized suc-avidin resin. Avidin is a highly basic glycoprotein composed of four identical subunits, each having a biotin-binding site. The use of this protein for the construction of affinity columns has the serious disadvantage that avidin binds nonspecifically to many components, especially negatively charged membrane proteins. This property can be eliminated by exhaustively succinoylating avidin with succinic anhydride to form suc-avidin.

[37] Synthesis of biotinyl derivatives of peptide hormones and other biological materials

Publisher Summary This chapter describes the procedures for modifying and labeling avidin and assessing the rate of dissociation of biotin derivatives from avidin. The use of avidin for the construction of affinity resins has serious disadvantages owing to the high content of basic amino acid residues that bind to anionic surfaces. However, conversion of the positively charged ɛ-amino groups of the lysine side chains to half succinoates, by reacting the protein with an excess of succinic anhydride, effectively eliminates this problem without markedly changing the biotin binding properties. The chapter explains that in preparing biotinylated ligands for avidin–biotin affinity columns, some consideration must be given to the nature of the interaction between the ligand and its binding protein or receptor. The biotin attachment site should be chosen so as not to interfere with ligand–receptor binding. Attachment of biotin to peptides or proteins may in some instances interfere with access of the biotinylated molecule to its avidin binding site and thus alter the strength of this interaction substantially.

Angiotensin stimulation of adrenal fasciculata cells

In this paper we provide evidence to show that the pathways by which adrenocorticotropic hormone (ACTH) and angiotensin II (AII) stimulate steroidogenesis in bovine fasciculata cells are only partially independent. Both hormones have the same intrinsic activity but a 500-fold higher dose of AII is required to achieve 50% stimulation of steroidogenesis. Whereas ACTH acts by way of cAMP, AII appears to operate through protein kinase C. The phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), and the calcium ionophore, A23187, each stimulate steroidogenesis and, when added together, act synergistically. To test the relationship between the ACTH and AII pathways, we added the two hormones simultaneously and measured steroid production. When the hormones were present at submaximal concentrations, their effects were additive. At maximal doses, steroid production was 40% above that elicited by either hormone alone. In contrast to the action of AII in the glomerulosa cell where it inhibits ACTH-stimulated cAMP formation, AII causes no inhibition in the fasciculata. Cycloheximide inhibits steroidogenesis stimulated by AII or a mixture of TPA and A23187. Scatchard analysis of the binding of 125I-AII to particulates from adrenal cortical fasciculata indicates the presence of a single class of binding sites (Kd = 0.6 X 10(-8) M). Binding is not inhibited by ACTH. Biotin-containing AII analogs that bind specifically to the particulates have been evaluated as potential tools for avidin-biotin affinity chromatography of the receptor. One of these, [N epsilon-6-(biotinylamido)hexyllys1, Val5] AII, is a promising candidate for receptor isolation.

The role of protein kinases in ACTH-stimulated steroidogenesis

Abstract The effect of highly purified bovine cytosolic adrenal cortical protein kinase isozyme II catalytic subunit (ATP: Protein Phosphotransferase EC 2.7.1.37) on the formation of pregnenolone from cholesterol in rat and bovine adrenal mitochondrial extracts has been investigated. No stimulation was observed although a low level incorporation of [ 32 P] from [ 32 P]-ATP into a component or components of the extract was detected. The mitochondrial extracts contained alkaline phosphatase activity that was inhibited by L-cysteine and dithiothreitol. It is concluded that the acute stimulation by ACTH of corticoid production in the rat adrenal does not involve protein kinase mediated phosphorylation of a component or components of the cholesterol sidechain cleavage mixed-function oxygenase system.

The steroidogenic activity of biotinylcorticotropin-avidin complexes.

The steroidogenic activity of complexes of [biocytin25]-corticotropin1–25 amide (biotinylcorticotropin) with avidin (1:1), (4:1) and (1:10) was compared to that of biotinylcorticotropin using isolated rat adrenal cells. Parallel log-dose responses and maximal stimulation were observed with all these materials. The 1:1 complex is approximately 25% as active as biotinylcorticotropin (ED5022.5 and 5.6 nM respectively). The 4:1 complex is more active than the 1:1 complex (ED509.0 nM). The presence of an excess of avidin (1:10 complex) does not interfere with the ability of biotinylcorticotropin to stimulate steroidogenesis (ED5018.0 nM). It is concluded that biotinylcorticotropin attached to avidin binds specifically to receptors on the rat adrenal cell and elicits its biological response. These results indicate that biotinylcorticotropin can be noninvasively labeled with 125I-avidin.

Bovine adrenal cortical protein kinases: isolation of the type II catalytic subunit.

Abstract Bovine adrenal cortical protein kinase type II catalytic subunit (ATP: Protein Phosphotransferase EC 2.7.1.37) has been purified by a method which relies on differences in net charge for the holoenzyme and the catalytic subunit. The purified subunit migrates as a single band on SDS disc gel electrophoresis (molecular weight, 43,500 daltons). The molecular weight based on gel filtration is 38,600. Isoelectric focusing resolves the subunit into 4 components all of which have the same pH optimum for activity. The apparent Km values for ATP are 24, 25, and 35 μM for the catalytic subunit, and the holoenzyme assayed in the absence or presence of cyclic AMP respectively; for histone, values of 0.9 and 1.0 mg/ml are obtained for the catalytic subunit and the holoenzyme. The pH-activity profile is broad with optimum activity at pH 6.5.

Bovine adrenocortical adenylate cyclase: Some properties of the solubilized, fluoride-activated enzyme

Abstract The activity of bovine adrenocortical plasma membrane adenylate cyclase can be maintained at 4°C in the presence of NaF. The half-life of the fluoride-stabilized enzyme is approximately 7 days. Maximal activation by fluoride requires approximately 20 min at 0°C and the level of activity attained is dependent on fluoride concentration. The enzyme from freshly harvested membranes can also be stimulated by ACTH 1 – 24 and Gpp(NH)p and the stimulatory effects of these two activators are additive. Prolonged exposure to either NaF or Gpp(NH)p precludes hormone activation. Optimal concentration for Gpp(NH)p activation is 10 −4 –10 −5 M . Treatment of the enzyme with a Tris-HCl buffer containing Lubrol-PX (1%), NaF, dithiothreitol, and MgSO 4 followed by sonication affords a preparation that does not sediment at 100 000 g in 1 hr. This material has a low specific activity; however, removal of the detergent on DEAE cellulose restores specific activity to its original level. A significant improvement in specific activity is observed following dialysis or ultrafiltration of the detergent-free 100 000- g supernatant. At this stage the enzyme can be lyophilized and stored at −70°C without loss of activity. The enzyme in the detergent-free 100 000- g supernatant behaves as a single peak that is included in Sepharose 6B. Comparison of the elution profile of the enzyme with profiles produced by a standard set of proteins suggests a molecular weight of 1 × 10 6 . Hydrophobic chromatography of the detergent-free 100 000- g supernatant on n -hexyl Sepharose 4B results in a fivefold enhancement of specific activity.