On the properties of porcine elastase released from its complex with human alpha-1-antitrypsin by alkaline cleavage.

These investigations were made to determine how the elastase released from its complex with alpha-1-antitrypsin at high pH is modified. Most of the elastase component precipitates on returning the pH to neutral. The elastase component and the native enzyme subjected to the same conditions of pH and temperature reacted to approximately the same extent with radio-actively labeled diisopropyl fluorophosphate. There were about two moles of dehydroalanine per mole of enzyme either in the presence or absence of complex formation. Thus, the enzyme is either still capable of reacting with diisopropyl fluorophosphate or it is denatured and thus inactivated by partial conversion of cystine residues to dehydroalanine. Anhydroelastase is apparently not formed during cleavage of the complex.     

Identification of rabbit microsomal cytochrome P-450 isozyme,form 1, as a hepatic progesterone 21-hydroxylase

Six highly purified forms of rabbit microsomal cytochrome P-450, isolated from hepatic microsomes, exhibit differences in the regiospecific metabolism of progesterone. Only one of the isozymes studied, form 1, catalyzes the formation of deoxycorticosterone from progesterone at an appreciable rate. This cytochrome P-450 isozyme may participate in the conversion of progesterone to deoxycorticosterone during pregnancy. All six forms of cytochrome P-450 catalyze 6β- and 16α-hydroxylation at the two concentrations of progesterone tested. Form 3b exhibits a lower apparent Km for 6β-hydroxylation than the other five.     

Isolation and partial characterization of a complex of several (pro-) endopeptidases from whole porcine pancreatic extract and from purified zymogen granules

Pancreatic zymogen granules contain exportable proteins at a very high concentration. The mechanism leading to this condensation is unknown. On the other hand, it is known that aqueous extract of pancreatic acetone powder precipitates at low ionic strength and acidic pH. The precipitable fraction is called ‘euglobulin’ in the literature. We thought that euglobulin could serve as a simplified model to study the condensation of some of the pancreatic exportable proteins. We have compared quantitatively and qualitatively the composition of euglobulins prepared from porcine pancreatic acetone powder and from lysates of purified pancreatic zymogen granules. They were found to be nearly identical, consisting of glycoprotein(s) and/or proteoglycan(s) associated to a proesterase activity, chymotrypsinogens C and D and proelastase. We conclude therefore, that the interactions between the constituents of euglobulin must be specific, since they can occur in the complex protein mixture of the whole organ to a similar extent as in the zymogen granules themselves. We have tried to identify the nature of these specific interactions. We were able to demonstrate that neither the granule membranes, nor the high-molecular-weight proteoglycan present in the granules (Reggio, H.A. and Palade, C.E. (1978) J. Cell. Biol. 77, 288–314) were responsible for the observed aggregation. Electrostatic interactions between acidic and basic proteins (Thomson, A. and Denniss, I.S. (1976) Biochim. Biophys. Acta 429, 581–590) were demonstrated between proelastase and chymotrypsinogens C and D. However, the possible roles of the glycoprotein(s) and/or proteoglycan(s) in the condensation process remain unknown.     

The stoichiometry of inhibition of human pancreatic elastase 2 by human alpha1-antitrypsin.

A 1:1 stoichiometry of inhibition of human pancreatic elastase 2 by human α₁-antitrypsin has been determined. The molar binding ratio was calculated using the results of a titration curve for elastase 2 inhibition by α₁-antitrypsin, an experimentally determined concentration of active sites in human elastase 2, and an extinction coefficient calculated from ultracentrifugation studies using interference optics.     

Preparation of concanavalin A free purified alpha-1-antitrypsin.

A simple technique is described to remove traces of concanavalin A (Con A) from human α-1-antitrypsin (α-1-AT) purified on commercially available Con A-Sepharose. The α-1-AT was fractionated from serum by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, Con A-Sepharose, and activated thiol-Sepharose at 4°C with solution pH ranges of 7.4–7.6 in all steps. Contaminating Con A was easily removed by binding α-1-AT through the reactive sulfhydryl group to the activated thiol-Sepharose gel and washing away the contaminating Con A with a solution of methyl-α-d-glucopyranoside before final elution of bound α-1-AT. This simple procedure yields purified α-1-AT free of traces of Con A. The α-1-AT was obtained in overall yields of 40–48% from serum with an average molecular weight of 53,500 ± 3000 determined on 15% disc polyacrylamide gels containing sodium dodecyl sulfate (SDS). The isolated α-1-AT exhibited unaltered Pi M phenotype compared to serum α-1-AT but contained traces of several other serum proteins.     

Alpha1-antitrypsin synthesis by human lymphocytes

$\text{De}$$\text{novo}$ synthesis of alpha₁-antitrypsin (α₁AT) by human peripheral lymphocytes has been demonstrated in the present study. Treatment of the mononuclear cells with concanavalin A(Con A) resulted in a triple increase in the amount of α₁AT synthesized by the untreated cells. A small amount of α₁AT, equivalent to that synthesized by the unstimulated mononuclear cells, was observed in cultures of monocyte-depleted lymphocytes, with or without Con A stimulation. Monocytes treated with or without Con A scarcely synthesized α₁AT. Conditioned media derived from monocyte enriched mononuclear cells treated with Con A enhanced about threefold α₁AT synthesis by the Con A-stimulated lymphocytes. α₁AT is suggested to be synthesized by lymphocytes assisted by monocytes.     

Amino acid sequence homology between the C3 chain of rat prostatic steroid binding protein and human alpha 2-macroglobulin

Using 32P-labeled DNA complementary to mouse submaxillary gland renin mRNA, we probed mRNA gel blots from mouse testis and kidney tissues. Poly(A)-RNA from testis contained a hybridizable RNA species which was blotted onto nitrocellulose paper. The molecular size of testicular renin mRNA (approximately 1600 nucleotides in length) was not significantly different from tht of kidney renin mRNA. Densitometric scan revealed that the content of renin mRNA in mouse testis was approximately 5-fold lower than that in mouse kidney. These results support the proposal that mouse testicular cells synthesize renin.     

Ankyrin is necessary for both drug-induced and ATP-induced shape change of human erythrocyte ghosts

Human erythrocyte membranes from ACD preseved blood were digested with trypsin (Protein: trypsin=100:1) and analyzed by SDS PAGE. After digestion for 20 sec at 0°C, only ankyrin disappeared but other bands including spectrin, band 4.1 and band 3 remained intact. In contrast to intact membranes, treatment with chlorpromazine or MgATP or HEPES did not induce shape change in these membranes. The number of transformable cells correlated closely with the amount of remaining ankyrin. We conclude that ankyrin is necessary for their shape change. This is the first direct evidence that ankyrin is involved in the maintenance of red cell shape. Three additional lines of indirect evidence are also presented.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

A simplified technique to isolate the porcine and human ileal intrinsic factor receptors and studies on their subunit structures.

The porcine intestinal intrinsic factor receptor was isolated with affinity chromatography utilizing vitamin B12-intrinsic factor-Sepharose and pH adjustments. The purification was about 70 000-fold and in sodium dodecyl sulphate electrophoresis it resolved into two carbohydrate-containing 70 000 and 130 000 dalton bands (alpha and beta subunits) indicating purity. The human receptor was similarly purified and radioiodinated for further studies. It was also composed of two subunits (90 000 and 140 000 dalton). The alpha subunits bound to anti-intrinsic factor antisera.     

Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.     

Induced changes in the affinity of 1,25-dihydroxyvitamin D3 receptors in chick intestine

The contents of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in plasma and intestinal mucose were increased by dietary calcium and by dietary phosphorus restriction. The concentration of intestinal occupied receptors for 1,25(OH)2D3 was higher in calcium-restricted birds. The affinity (association constant) of intestinal receptors for 1,25(OH)2D3 was lower in phosphorus-restricted chicks, as compared to control or calcium-restricted chicks. The number of binding sites were not influenced by dietary calcium or phosphorus restriction.