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.     

Sialic acid activation.

Cytidine 5'-monophosphosialic acid (CMP-sialic acid) is the activated form of sialic acid which is required for the biosynthesis of sialic acid-containing complex carbohydrates. Its discovery over 30 years ago by the laboratory of Dr Saul Roseman was a landmark in research dealing with the biosynthesis of these compounds. A review is presented of the salient features concerning this molecule: its discovery, chemistry, biosynthesis, subcellular location, enzymatic cleavage, transport and molecular biology. This report does not deal with its utilization by the sialyltransferases.     

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.     

B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis

Considerable evidence indicates that cross-linking of B cell surface Ig results in a "first signal" in B cell activation. We have shown that transduction of this signal is manifest by changes in plasma membrane potential leading to increased expression of surface I-A antigen. In previous studies, we have provided evidence that suggests that this signal is transduced via phosphatidylinositol (PI) hydrolysis liberating diacylglycerol (DAG), which subsequently activates protein kinase C. These biochemical events are aspects of a transmembrane signal transduction mechanism that is common in nature and utilizes the PI metabolic cycle for generation of "second messenger" diacylglycerol. Here we report direct evidence that treatment of B cells with various antibodies to surface Ig results in activation of the PI cycle. Results suggest that the increased phospholipid metabolism that occurs in B cells in response to anti-Ig involves only those phospholipids in the PI cycle and is a consequence of turnover of existing lipid rather than de novo synthesis. Furthermore, we show that PI cycle activation requires cross-linking of membrane Ig and is inhibitable by increased intracellular cyclic AMP. These findings are particularly important in view of previous studies that have shown identical requirements for and inhibitability of induction of B cell membrane depolarization and increased I-A expression. Thus, these results are consistent with our previous hypothesis that early B cell activation events initiated by receptor Ig occupancy are mediated via PI hydrolysis, diacylglycerol generation, and protein kinase C activation.     

Surface activation of pro-cathepsin L.

Pro-cathepsin L is an inactive zymogen that has been shown previously to undergo autolysis at pH 3.0 to give mature forms of the enzyme. We have now been able to demonstrate that this enzyme can undergo activation at pH 5.5 in the presence of negatively charged surfaces. Activation could also be measured at pH 6.0, but no activation occurred at pH 6.5 or higher. The initiation of activation depends upon the presence of a small percentage of active pro-enzyme, and this is then followed by a more rapid activation to give mature forms of the enzyme. No significant intermediate molecular forms of the enzyme were seen. The time taken for processing of the pro-enzyme to single-chain mature enzyme is comparable to that seen in biosynthetic pulse-chase experiments.     

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

Pristane induced gene activation.

Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

Connective tissue activation. XXI

Summary The quantitative radiochemical methodology described in this report allows a major increase in information generation, increased experimental flexibility, improved statistical control, and increased diversity of information per culture. Other advantages relate to economies of technical time, supplies, cells, and test materials per individual culture. Microcultures of human synovial cells incorporate [¹⁴C]glucosamine into hyaluronic acid that accumulated primarily in the media and to a lesser extent in the cell mass. CTAP-I (from lymphoid cells), CTAP-III (from human platelets), PGE₂, dibutyryl cAMP, and poly(I)·poly(C) markedly stimulated hyaluronate synthesis, whereas cortisol, cycloheximide, and tunicamycin inhibited stimulated synthesis. Time studies with cycloheximide indicated that translation, essential for the activation of synovial cells, was completed by 17 h postexposure to CTAP-I. Tunicamycin also seemed to inhibit CTAP-I induced activation primarily by interpering with translation; however, tunicamycin also caused modest post-translational inhibition of hyaluronate synthesis in activated adult human synovial cells. Study supported by U.S. Public Health Service Grant AM-10728, the Michigan Chapter of the Arthritis Foundation, and Michigan Memorial-Phoenix Project Grant No. 517.     

Monovalent cation activation of tryptophanase.

The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.     

GTPase-mediated activation of ATP sulfurylase.

GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase.     

Prorenin and gene activation.

After the discovery of an inactive, putative renin precursor that could be proteolytically activated, and the proteases involved in vivo, Morris and co-workers directly demonstrated that renin is indeed synthesized as a "pro" form, and from genetic coding sequences they provided the structure of human prorenin. The gene is inactive and must be activated in prorenin-synthesizing tissues. To study the mechanism involved, we have performed transient expression analyses of putative regulatory DNA of the human gene (REN). 5'-Flanking DNA, extending from residue -144 to -2400, was linked to a reporter gene, viz. that for chloramphenicol acetyl transferase (CAT), and its ability to drive a heterologous (thymidine kinase, tk) promoter was examined by transfecting plasmid constructs into cells in culture and measuring CAT activity 48 h later. Because suitable renin-synthesizing cells were not available, choriocarcinoma (JEG-3) and cervical carcinoma (HeLa) cells were used. Although this DNA caused a reduction in CAT activity relative to the positive control, examination of a range of subfragments suggested that the -2400 to -144 region did not contain negative regulatory elements. In contrast, all fragments containing the -149 to +13 DNA segment gave CAT activities that were lower than the promoterless control. Together, the data were consistent with the presence of negative regulatory element(s) in that fragment of DNA that contained the REN promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress stimuli-induced lymphocyte activation.

Oxidants, heavy metals, and heat shock, collectively known as stress stimuli, induce the synthesis of a variety of proteins, termed stress proteins, and enhance glucose uptake. In this study, we have demonstrated that stress stimuli enhance protein tyrosine phosphorylation (PTyr-P), modulate protein tyrosine phosphatase (PTPase) activity, activate the src family protein tyrosine kinase (PTK), p56lck, and enhance glucose uptake in human peripheral blood mononuclear cells. The heavy metal Hg2+ and heat shock stimulated PTPase activity at an optimal dose, whereas the oxidant phenylarsine oxide (PAO) was only marginally stimulatory. Treatment of lymphocytes with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stress stimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and p56lck activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PAO were effective in activating PTPase than those effective in eliciting p56lck activation. We could not demonstrate a correlation between an effective dose inducing PTyr-P and glucose uptake. Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p56lck, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or preventing dephosphorylation of target proteins which are involved in the biological response. Our data may also provide the biochemical basis for the previously reported mitogenic effects of Hg2+ on lymphocytes.     

Selective phospholipase C activation.

Phospholipase C is a family of cellular proteins believed to play a significant role in the intracellular signaling mechanisms utilized by diverse hormones. One class of hormones, polypeptide growth factors, elicits its influence on cellular function through stimulation of cell surface receptor tyrosine kinase activity. Certain growth factors appear to stimulate cellular phospholipase C activity by selective, receptor-mediated tyrosine phosphorylation of the phospholipase C-gamma 1 isozyme. While the role of phospholipase C activity in growth factor regulation of cell proliferation remains to be clarified, the selective growth factor-stimulated tyrosine phosphorylation and activation of phospholipase C-gamma 1 is an interesting example of enzyme-substrate interaction at the crossroads of two important intracellular signaling pathways.