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.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Structural studies on epsilon-prototoxin of Clostridium perfringens type D. Localization of the site of tryptic scission necessary for activation to epsilon-toxin.

The mechanism of activation of ε-prototoxin to ε-toxin has been ascertained from partial amino acid sequences of both ε-prototoxin and ε-toxin. The activation of ε-prototoxin from $\text{Clostridium perfringens}$ type D by brief exposure to trypsin is caused by scission of a peptide bond between Lys₁₄ - Ala₁₅. A small peptide (14 amino acid residues) is split from the NH₂-terminus of the ε-prototoxin to give the active ε-toxin.     

Subunits of yeast RNA polymerase I involved in interactions with DNA and nucleotides.

Ovine mammary gland mRNAs were translated in a wheat-germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on α-lactalbumin isolated by immunoprecipitation from the mixture of radiolabelled lactoproteins showed the occurrence of an hydrophobic 19 residues long amino terminal extension. The pre-protein represents the primary translation product since the amino terminal methionyl residue was found to be donated by initiator $\text{Met-tRNA}{}_{\mathrm{i}}{}^{\mathrm{Met}}$. Comparison of the signals of ovine α-lactalbumin and hen's egg white lysozyme, two homologous proteins which are thought to be derived from a common ancestor, suggests that the signal region has evolved at least as rapidly as the remaining part of the polypeptide chain.     

The amino terminal sequences of acid proteases-human pepsin and gastricsin and the protease of Rhizopus chinensis.

The amino acid sequences near the amino termini of human pepsin (34 residues) and gastricsin (24 residues) and the acid protease from $\text{Rhizopus chinensis}$ (27 residues) have been determined using automated Edman degradation. From these results three additional observations were made. First, two structural variants have been observed for human gastricsin and for the $\text{Rhizopus}$ protease. Both cases are apparently genetic in origin. Second, a stretch of sequence in the $\text{Rhizopus}$ protease, residues 14 to 26, is highly homologous to the known sequence of porcine pepsin at the region of residues 11 to 23. Third, the sequences of the NH₂-terminal region of human pepsin and gastrisin are homologous.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Cell-free synthesis of angler fish preproinsulin: Complete amino acid sequence of the signal peptide

Messenger ribonucleic acid isolated from angler fish ($\text{Lophius}\text{americanus}$) islets of Langerhans was translated in the wheat germ cell-free protein synthesizing system containing different combinations of radioactive amino acids. Preproinsulin (～ 11,000 daltons) was identified amongst the translation products, by sodium dodecyl sulfate gel electrophoresis, and subjected to microsequencing techniques. The fish preproinsulin was found to possess an NH₂-terminal signal peptide of 24 amino acids, with regions of homology to human, rat and chicken preproinsulin signal sequences.     

Maturation of collagenous tissue: Specific invivo proteolytic cleavage of only α1(I) chains

A partially cleaved α1(I) chain, α1_χ, has been isolated from earlier synthesized or older (acid-extracted) guinea pig skin collagen. The α1_χ component is shown to be absent from the newly synthesized (neutral salt-extracted) collagen. This degradation is a result of specific $\text{in}\text{vivo}$ proteolytic sission of α1(I) chain since the soluble collagen has no corresponding product from the α2 chain. The $\text{in}\text{vivo}$ proteolytic cleavage is believed to result from processes related to natural physiological maturation of collagenous tissue.     

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Synthesis of α,ω-bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands and complexes of rhodium(I)

α,ω-Bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands can be prepared in very high yields via reaction of the appropriate dihalide with two equivalents of LiPPh₂. For the [Rh(COD)($\text{P P}$)][ClO₄] complexes of these ligands, the $\text{P P}$ ligands with five or less atoms in the alkane or ether bridge form monomeric complexes via η²-coordination. In general the ligands with eight or more atoms in the bridge give di- or polynuclear species. In addition the long chain diphosphino-polyethers form – to a small extent – monomeric species by η²-coordination.     

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me₂SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of $\text{α}\text{β}\text{-}\text{mRNA}$ is maintained during induction. In maximally induced cells, the $\text{α}\text{β}\text{-}\text{globin}$ mRNA ratios are approximately 1 in cells induced by Me₂SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.     

5-acyloxyoxazoles as serine protease inhibitors. Rapid inactivation of thrombin in contrast to plasmin.

Thrombin is rapidly inactivated by affinity labeling with 5-acyloxyoxazoles ($\text{I}$). The reaction with 2-phenyl-4-(3-N-nitroguanidinopropyl)-5-pivaloyloxyoxazole ($\text{Ia}$) proceeds via a hydrophobic binding step to give inactive pivaloyl thrombin, which slowly but fully reactivates under mildly alkaline conditions. Trypsin and chymotrypsin are also inactivated by $\text{Ia}$. In contrast, plasmin is very slowly affected by high concentrations of $\text{Ia}$ that would inhibit thrombin almost instantaneously. These observations correlate with the selectivity of proflavin binding to these enzymes. The 2-methyl analog of $\text{Ia}$ shows poor binding to thrombin and consequently inactivates it much more slowly than does $\text{Ia}$.     

Altered adenosine triphosphatase activities of natural actomyosin from rat hearts perfused with isoprenaline and ouabain

Perfused rat hearts were treated with isoprenaline (10^?6M) or ouabain (5.5 × 10^?6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with ³²P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg²⁺-ATPase activities, at saturating Ca²⁺ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the V_max[Ca²⁺] of this ATPase activity. The Ca²⁺ sensitivity of the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases.Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca²⁺-ATPase activities. In addition, the V_max[ATP] of the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca²⁺ sensitivity of $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ activity.We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca²⁺, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca²⁺ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca²⁺-ATPase activity, as well as on the rate of cross-bridge formation.     

A peptide released from plasma fibrin stabilzing factor in the conversion to the active enzyme by thrombin

A peptide, which was released accompanying with the activation of bovine plasma fibrin stabilizing factor (FSF) by thrombin, was isolated and characterized. The peptide consisted of Asp4, Thr₃, Ser₄, Glu₄, Pro₅, Gly₄, Ala₄, Val₂, Ile₁, Leu₂, Phe₁, and Arg₃. The content of proline was highest in all of these amino acids. The carboxyl-terminal residue of the peptide was identified as arginine. However, no N-terminal amino acid reactive with phenyl$\text{iso}$thiocyanate and dansyl chloride could be determined. Edman degradation on the inactive FSF showed glutamic acid or glutamine as one N-terminal residue. After the activation of FSF by thrombin, glycine was identified as a second N-terminal residue, in addition to glutamic acid (glutamine).These results indicate that the transformation of FSF to the active enzyme by thrombin involves proteolysis of an arginyl-glycyl bond located in the N-terminal region of one of the subunits of the proenzyme.     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

Molecular characteristics of bovine factor X activated in concentrated sodium citrate solution

Preparations of the zymogen form of bovine factor X were incubated in 25% $\text{w}\text{v}$ sodium citrate at room temperature. The rate of activation of factor X was dependent on the extent of contamination with factor VII, prothrombin, and thrombin. The activated factor X was isolated by DEAE-cellulose chromatography. Analysis of the final product by sedimentation velocity centrifugation coupled with measurements of the rate of boundary spreading, high-speed sedimentation equilibrium, and gel filtration chromatography provided evidence for a single molecular species undergoing reversible association-dissociation with a monomeric molecular weight of 48,000. In the absence of mercaptoethanol a single band was seen by disc electrophoresis and by SDS-acrylamide electrophoresis but after disulfide reduction two components of molecular weights 30,000 and 17,000 were visible. The protein contained large amounts of acidic amino acids but no carbohydrate. The N-terminal amino acids were alanine and isoleucine and 1 mole C-terminal arginine per mole protein was found. These characteristics are very similar to those of factor X activated with Russell's viper venom.When a BaSO₄ eluate of bovine plasma rich in prothrombin was allowed to stand in 25% sodium citrate both thrombin and activated factor X were generated. Chromatography of the isolated activated factor X on Sephadex G-200 as well as disc electrophoresis showed that it behaved identically with the enzyme obtained from purified zymogen and was clearly distinguishable from autoprothrombin c, a glycoprotein possessing qualitatively similar biological activity (Seegers, W. H., Cole, E. R., Harmison, C. R., and Marciniak, E. (1963) Can. J. Biochem. Physiol.41, 1047).