Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α₂-macroglobulin. On chromatography on Sephadex G-200, α₂-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α₂-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α ₂ ⁻ acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α₂-macroglobulin as well as α₂-macroglobulin bound proteases, inactivating them.     

Binding of alpha 2-macroglobulin and haptoglobin to Actinomyces pyogenes

All 25 cultures of Actinomyces pyogenes tested in the present study bound 125I-labelled human alpha 2-macroglobulin with a mean binding of 65.6%. Thirteen cultures also bound 125I-labelled human haptoglobin with a mean of 51.5%. None interacted with fibrinogen, fibronectin, immunoglobulin G, or albumin. Twenty-eight cultures representing other species of actinomycetaceae did not show any interaction with alpha 2-macroglobulin, haptoglobin, and other plasma proteins tested. The binding of alpha 2-macroglobulin and haptoglobin to A. pyogenes was saturable and could be completely inhibited by the respective unlabelled plasma proteins. The binding of alpha 2-macroglobulin could not be inhibited by unlabelled haptoglobin. On the other hand, alpha 2-macroglobulin blocked the binding of haptoglobin, possibly by steric hindrance. Treatment of the bacteria with trypsin reduced their binding activities for alpha 2-macroglobulin and haptoglobin indicating the protein nature of the binding sites. Exposure to heat (1 h, 80 degrees C) significantly diminished the binding activity for haptoglobin, but not that for alpha 2-macroglobulin. The binding of alpha 2-macroglobulin and haptoglobin could be an important feature in the classification of A. pyogenes among the members of actinomycetaceae.     

A solid-phase immunosorbent assay to determine the proteinase binding capacity of alpha 2-macroglobulin using 125I-trypsin as indicator proteinase

Hyperimmune sera against human alpha 2 macroglobulin were raised in rabbits following immunization with 's' alpha 2-macroglobulin over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for 's' and 'f' alpha 2-macroglobulin (plasma alpha 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4 X 10(-6) as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, 's' alpha 2-macroglobulin and both 'f' alpha 2-macroglobulins (27 and 82% saturation of 's' alpha 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact alpha 2-macroglobulin subunit (Mr 182000) with 125I-labeled trypsin binding capacity of immunoimmobilized alpha 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between 's' alpha 2-macroglobulin and 'f' alpha 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol alpha 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized 's' alpha 2-macroglobulin with trypsin could be accomplished by incubation with a 100-200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol alpha 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized alpha 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to alpha 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of alpha 2-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive alpha 2-macroglobulin of 0-53% when compared to the trypsin-binding capacity of normal plasma alpha 2-macroglobulin.     

Human leucocyte neutral proteases, with special reference to collagen metabolism.

Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.     

The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.     

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.     

Plasma proteinase inhibitors in Morris hepatoma-bearing rats: changes in the blood level and synthesis in tissue slices

The antiproteinase activities against trypsin, chymotrypsin, elastase, papain and rat leucocyte proteinases were determined in plasma from control and Morris hepatoma-bearing rats. Bovine trypsin and chymotrypsin were similarly inhibited by the two types of plasma whereas porcine pancreatic elastase, papain and rat leucocyte neutral proteinases were more efficiently inhibited by plasma from tumour-bearing rats. The increased plasma concentrations of some proteinase inhibitors, as determined by rocket immunoelectrophoresis, are suggested to be responsible for the observed differences in inhibition. The highest increases in plasma of tumour-bearing rats were observed for alpha 2-macroglobulin and alpha 1-acute-phase globulin. The synthesis and secretion of six proteinase inhibitors: antithrombin III, alpha 1-proteinase inhibitor, alpha 1-macroglobulin, alpha 2-macroglobulin, alpha 1-acute-phase globulin and haptoglobin, as well as albumin, were measured in tissue slices from rat liver and Morris hepatoma after incubation with [14C]leucine. Local inflammation inflicted upon the tumour-bearing rats increased formation of acute-phase proteins in liver slices but not in hepatoma slices.     

Inactivation of alpha(2)-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active alpha(2)- macroglobulin (80 mug/ml) totally inhibits the proteolytic activity of trypsin (14 mug/ml) by trapping this protease. But after a 20 min incubation of alpha(2)-macroglobulin at 37 degrees C with 2 x 10(6) human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10(-7) M) and cytochalasin B (10(-8) M), 100% of trypsin activity was recovered, indicating a total inactivation of alpha(2)-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates alpha(2)-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10(-7) M also inactivates alpha(2)-macroglobulin, which indicates that an important cause of alpha(2)-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.     

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.     

A redetermination of the concentration of alpha 2-macroglobulin in human plasma

The concentration of alpha 2-macroglobulin in human plasma has been remeasured utilizing a carefully isolated and characterized sample of alpha 2-macroglobulin as a standard. A highly purified sample of alpha 2-macroglobulin with a total trypsin binding capacity of 1.7 mol trypsin/mol alpha 2-macroglobulin was used as a standard for both a radial immunodiffusion and a rocket immunoelectrophoresis technique. With this preparation as a standard, the concentration of alpha 2-macroglobulin in a normal plasma pool over 10,000 donors was found to be about 1.2 mg/ml. A similar concentration (1.3 mg/ml) was found when using a functional trypsin binding assay. This concentration is considerably less than the usually accepted mean of the normal range for alpha 2-macroglobulin.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Modulation of antigen-induced T cell proliferation by alpha 2M-trypsin complexes

Proteinase-complexed alpha 2-macroglobulin (alpha 2M) could be shown to interfere with T cell proliferation in response to antigen presented by autologous antigen-pulsed monocytes (M phi) (antigen-induced M phi-T cell interaction, MTI). Addition of alpha 2M-trypsin (alpha 2M X T) complexes to cultures of T cells and antigen-pulsed M phi led to a dose-dependent decrease of T cell proliferation (up to 91% inhibition of the T cell response), whereas the same concentrations of free (native) alpha 2M had no effect on antigen-induced MTI. The observed interference with MTI could be attributed to residual enzymic activity of the alpha 2M X T complex. Addition of aprotinin, a low Mr protein proteinase inhibitor able to penetrate to the enzyme entrapped within the alpha 2M molecule and thus bind to and inactivate the enzyme's active site, resulted in a reversal of the alpha 2M X T-induced biological effect. Inactivation of the enzyme's active site within alpha 2M X T was monitored by a decrease in the hydrolytic activity of the complex. Kinetic studies (addition of alpha 2M X T 24 to 48 hr after culture onset was shown to be still inhibitory) indicated an effect at the level of the T cell or its mediators, but an overnight incubation of T cells with alpha 2M X T did not alter these cells' capacity to proliferate in response to an antigenic stimulus. An additional effect of alpha 2M X T on the antigen-presenting cell cannot be ruled out at present. However, alpha 2M X T did not alter the percentage of monocytes expressing HLA-DR, -DP, and -DQ or interfere with interleukin 1 release if added to M phi at concentrations that significantly inhibited MTI. Furthermore, incubation of M phi with alpha 2M X T for 1 hr before antigen pulsing had no effect on the M phi antigen presenting capacity.     

Glycemic control reverses increases in urinary excretions of immunoglobulin G and ceruloplasmin in type 2 diabetic patients with normoalbuminuria.

To examine whether urinary excretions of plasma proteins with molecular radii of 45-55 A and different isoelectric points such as IgG (pI = 7.4) and ceruloplasmin (pI = 4.4) increase selectively in normoalbuminuric type 2 diabetic patients, urinary albumin excretion rate (AER), renal clearances of IgG, ceruloplasmin and alpha2-macroglobulin, and creatinine clearance (Ccr) were studied in timed overnight urine samples of 36 diabetic outpatients and 16 control subjects. Furthermore, to examine effect of glycemic control on these urinary protein excretions, the same analysis was performed before and after glycemic control in 17 diabetic inpatients admitted for glycemic control. Renal clearances of IgG and ceruloplasmin were significantly higher in diabetic outpatients than in the control group, whereas AER and renal clearance of alpha2-macroglobulin did not differ. Glycemic control caused significant decreases in renal clearances of IgG and ceruloplasmin, accompanied with tendency for Ccr to decrease (p = 0.055). The present results, together with our previous finding of selectively increased urinary excretions of 45-55 A sized plasma proteins in parallel with enhanced glomerular filtration rate after acute protein loading, led us to conclude that enhanced intraglomerular hydraulic pressure may cause increases in clearances of IgG and ceruloplasmin, and that this change can be reversed by strict glycemic control in normoalbuminuric diabetic patients.     

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.     

Effect of cobra and viper venoms on alpha 2-macroglobulin activity in human, bovine, and goat sera

Incubation of human serum with cobra or viper venoms (10 micrograms/0.1 ml serum) caused negligible decrease in total protease inhibitory activity whereas alpha 2-macroglobulin activity was reduced by 67.0-82.0% in 16 hr. The action of venoms on MG activity was time dependent. Human alpha 2-macroglobulin activity was reduced to a much greater extent than goat or bovine factors by the venoms. While 25 micrograms venoms/0.1 ml serum caused 60-100% inhibition of human alpha 2-macroglobulin activity, the bovine factor was not affected under similar conditions. Goat alpha 2-macroglobulin was affected to the extent of 0-20%. Evidence is provided to show that venom proteases generate endogenous proteases in situ in human plasma or serum which in turn bind to alpha 2-macroglobulin. The venom-mediated action was abolished by prior dialysis of the serum or its dilution. Ethylenediaminetetraacetate at 10(-3) M concentration also blocked the reaction. While phenylmethylsulfonyl fluoride had no effect, pepstatin in the concentration range 10(-2) to 10(-3) M caused partial inhibition of the venom-mediated inhibition of alpha 2-macroglobulin activity in human serum.     

Lactate, alanine and glutamine metabolism in isolated canine pup liver cells

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.     

A case of ruptured hepatoma followed by elastase-induced disseminated intravascular coagulation

In the present study, the first case of ruptured hepatoma followed by disseminated intravascular coagulation is reported. An elastase-like enzyme which possessed elastolytic and caseinolytic activities was confirmed from patient plasma. On the other hand, no elastase activity was detected in the plasma of patients with hepatitis, liver cirrhosis or hepatoma without disseminated intravascular coagulation. The patient plasma did not possess H-D-Val-Leu-Lys-p-nitroanilide hydrochloride, succinyl-L-alanyl-L-alanyl-p-nitroanilide, and pyro-Glu-Pro-Val-p-nitroanilide amidolytic activities. However, when chromatographed on Sephadex G-200, the presence of low-molecular weight plasminogen was confirmed. Its molecular weight was approximately 52,000. A slight decrease of alpha 2-plasmin inhibitor was noted, but no decrease of alpha 2-macroglobulin was detected.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of rat alpha 1-macroglobulin. Identification of an intracellular precursor

Alpha 1-macroglobulin was purified from rat plasma by gel filtration (Sephacryl S-300) and ion exchange chromatography (DE52). Analysis of the purified alpha 1-macroglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides: a light chain which could be resolved into a double band (36/38 kDa) and a heavy chain (160 kDa). Under non-reducing conditions complexes of 200 and 400 kDa could be demonstrated. Antibodies were raised against both chains of alpha 1-macroglobulin which did not cross-react with either rat alpha 2-macroglobulin or rat alpha 1-inhibitor 3. It was shown that in the medium of [35S]methionine-labeled hepatocytes the two subunits of alpha 1-macroglobulin are linked by disulfide bridges. Intracellularly, however, a high molecular mass polypeptide (185 kDa) could be immunoprecipitated with either the antiserum to the heavy or the light chain of alpha 1-macroglobulin, indicating the existence of a polyprotein precursor. Also in a cell-free translation system alpha 1-macroglobulin was synthesized as a polyprotein consisting of heavy and light chains (162 kDa). In a pulse-chase experiment using tunicamycin to block N-glycosylation, alpha 1-macroglobulin secretion was totally inhibited. This finding reflects the importance of the oligosaccharide side chains for the proteolytic processing to the two subunits and/or secretion of alpha 1-macroglobulin.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.