Structural alterations in alpha1-antichymotrypsin from normal and acute phase human plasma

Human alpha₁-antichymotrypsin, isolated at pH 8.0 from both normal and acute phase plasma, has been found to have two different amino terminal sequences despite the fact that inhibitory activities are unchanged. In normal plasma over 90% of the protein has an amino terminal sequence beginning with aspartic acid and less than 10% with arginine. However, in acute rheumatoid arthritis plasma 55% of the inhibitor begins with arginine and the remainder with aspartic acid. Sequence studies indicate that a fifteen amino acid peptide fragment has been cleaved to yield the arginine protein. Human alpha₁-proteinase inhibitor also shows this heterogeneity, but the ratios do not change between normal and acute phase plasma. It may well be that the missing peptide has some biological activity manifested only in the acute phase state.     

Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides

A highly purified and active cytochrome $\text{b}$-$\text{c}$₁ complex has been isolated from the chromatophores of the photosynthetic bacteria $\text{Rhodopseudomonas}\text{sphaeroides}$ R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome $\text{c}$ with a turnover number of 1500 per minute at 23° based on cytochrome $\text{c}$₁. It contains 8.3 nmoles of cytochromes $\text{b}$ and $\text{c}$₁ per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Purification and crystallization of a protein complex from Bacillus stearothermophilus ribosomes

A complex of ribosomal proteins B-L8 and B-L13 from Bacillus stearothermophilus has been isolated and crystallized. The cell dimensions are $\text{a = b = 108}\text{A}\text{?}\text{, c = 242}\text{A}\text{?}$ with the space group P4₁2₁2 (or P4₃2₁2). The asymmetric unit probably contains two complexes with a 4:1 stoichiometry of the proteins.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Role of the plasma membrane in the mechanism of cholesterol efflux from cells

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [³H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu₅AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for plasma membrane vesicles of Fu₅AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu₅AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu₅AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for cholesterol efflux from these cell lines.     

Isolation and characterization of isoprothrombin in the rat

Isoprothrombin, a protein antigenically related to prothrombin, which accumulates in the hepatic endoplasmic reticulum of vitamin K-deficient or warfarin-treated rats, has been isolated by affinity chromatography employing rat prothrombin antibodies linked to Sepharose. Isoprothrombin has the same molecular weight as prothrombin but a different mobility on disc gel electrophoresis, is not barium adsorbable nor activatable to thrombin by factor X_a. Isoprothrombin is converted to thrombin by $\text{Echis carinatus}$ venom through the same intermediates as prothrombin.     

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.     

Microcalorimetric study of glutamine fixation on the glutamine-binding protein of Escherichia coli.

The enthalpy variation (ΔH) induced by addition of glutamine to glutamine binding protein isolated from $\text{E. coli}$ has been studied by microcalorimetry. The reaction was very exothermic. The free energy variation (ΔG) was calculated from the dissociation constant (K_D) measured by dialysis techniques. The entropic variation (ΔS) was deduced from ΔG and ΔH values; it was found highly negative, indicating that an important conformational change is occuring. Comparison with others binding proteins and possible significance of such a phenomenon is discussed.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

alpha 1-Adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin.

Binding of (³H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (³H)-prazosin bound/mg protein and the K_D was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (³H)-prazosin as follows: (?)adrenaline > (?)noradrenaline > (?)phenylephrine ? ($\text{+}$)isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ? piperoxan > yohimbine > sulpiride > propranolol. The K_D for beta-adrenoceptors assessed by (?³H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (³H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha₁) adrenoceptors in myocardial tissue.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Shape complementarity at the hemoglobin alpha 1 beta 1 subunit interface

A computational method for attempting to predict protein complexes from the coordinates of the individual proteins has been developed. It is based on matching complementary patterns of knobs and holes. The computer algorithm correctly and uniquely predicts the association of the alpha and beta subunits to form the αβ dimer corresponding to the α₁β₁ interface in the hemoglobin tetramer. It fails to correctly dock trypsin inhibitor onto trypsin. Nevertheless, this lone success is still a significant advance over previous protein-docking algorithms. The method is also important because it introduces several ways to measure the shape of protein surface regions.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Preliminary x-ray diffraction studies on the "goose-type" lysozyme from the egg-white of the black swan Cygnus atratus

The “goose-type” lysozyme isolated from the egg-white of the black swan Cygnus atratus has been crystallized. The space group is P2₁ with one molecule of protein in the asymmetric unit. The cell parameters are $\text{a = 46.2}\text{A}\text{?}\text{, b = 65.1}\text{A}\text{?}\text{, c = 38.7}\text{A}\text{?}$, β = 110 °. The crystals diffract to a resolution of 2.25 Å and a set of diffraction data for the native protein has been collected photographically.     

Isolation and properties of oxidized alpha-1-proteinase inhibitor from human rheumatoid synovial fluid

Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P₁ reactive site and the other at P₈, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed $\text{in vivo}$, presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur.     

P1-purinoceptor antagonism by clonidine in the rat caecum

Adenosine as well as 2-chloroadenosine induced concentration-dependent relaxation of the rat caecum. Although clonidine (10^?9?10?⁶M) did not exhibit any effect $\text{per se}$ yet it shifted the concentration-response curve for adenosine as well as 2-chloroadenosine to the right in a parallel fashion without diminishing the maximal response. The pA₂ value for the interaction of clonidine with adenosine was found to be 8.71 (slope 0.92). Guanfacine, another alpha₂ agonist, did not modify the adenosine-, 2-chloroadenosine-induced relaxant effects. These observations indicated that clonidine blocks P₁-purinoceptors competitively without any involvement of alpha₂-receptors.     

An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Separation and characterization of two bovine alpha1-fetoprotein molecular variants by concanavalin A-Sepharose chromatography.

Two molecular variants of bovine alpha₁-fetoprotein were separated by affinity chromatography of fetal calf serum on a concanavalin A-Sepharose column. Radialimmunodiffusion assay of bovine alpha₁-fetoprotein revealed that 29% of the alpha₁-fetoprotein in fetal serum lacked concanavalin A-binding activity whilst 71% of the alpha₁-fetoprotein was capable of binding to the lectin. These two bovine alpha₁-fetoprotein variants show antigenic identity suggesting that the polypeptide chain rather than the carbohydrate moiety of the alpha₁-fetoprotein molecule is the antigenic determinant.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.