Biochemical studies on a low molecular weight antiplasmin of human blood.

We previously reported a low molecular weight antiplasmin present in human platelets and plasma. This material as isolated by ultrafiltration was heterogeneous. The fraction showing maximum inhibition has been further purified by countercurrent distribution. The N-terminal residue of this purified inhibitor is alanine. Amino acid analysis showed glycine to be the predominant residue, and no basic amino acid was detected. Kinetic studies suggested a competitive inhibition of plasmin via enzyme-inhibitor complex formation. Carbohydrate and phosphate could not be detected. These data were similar for both platelet antiplasmin and serum antiplasmin. The relationship between the platelet and the serum inhibitor remains unclear. This low molecular weight antiplasmin may contribute to the consolidation of the thrombus.     

mRNA for the plasminogen activator of the urokinase type: translation in oocytes of Xenopus laevis

Poly(A)+-RNA from human kidney and human embryonal lung fibroblasts fractionated by sucrose gradient centrifugation was translated in Xenopus oocytes. Assay for plasminogen-dependent fibrinolytic activity detected synthesis of secreted plasminogen activator and revealed the active fraction of poly(A)+-RNA with a sedimentation coefficient of approximately 23S. Translation products of the active fraction were immunoadsorbed by antiurokinase monoclonal antibodies immobilized on sepharose. Gel electrophoretic analysis of the protein products showed that the 23S fraction of poly(A)+-RNA from human kidney contains mRNA for single-chain urokinase-type plasminogen activator with apparent molecular weight of approximately 50 kDa.     

Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.     

The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

Identification of a calcium-independent phospholipase A2 in rat lung cytosol and differentiation from acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether)

The 100 000 X g supernatant of total rat lung homogenate was found to contain at least three phospholipase A2-type activities. Gel filtration separated a low molecular weight and Ca2+-requiring phospholipase A2 from Ca2+-independent acylhydrolase peak with an apparent higher molecular weight. Upon DEAE-cellulose chromatography this fraction was separated into a Ca2+-independent acylhydrolase and a Ca2+-independent platelet-activating factor-acetylhydrolase with no apparent overlap in acyl chain length specificity. The long-chain acylhydrolase was shown to exhibit specificity for the ester bond at the sn-2-position. Ca2+-independent phospholipase A2 activity was inhibited by p-bromophenacylbromide and was resistant to diisopropylfluorophosphate. In contrast, the Ca2+-independent acetylhydrolase activity was inhibited by diisopropylfluorophosphate but was unaffected by p-bromophenacylbromide.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.     

Comparative characteristics of tRNA-methyltransferases from rabbit liver and myocardium under normal conditions and in experimental ischemia

Comparative studies of the state of aggregation and activity of tRNA-methyltransferases in cytosol (105,000 X g supernatant) from normal and ischemic rabbit liver and myocardium were carried out. The optimal conditions (pH, protein concentration, ionic composition of incubation mixture) for the determination of activity of tRNA-methyltransferases were elaborated. The protein fraction precipitated at 55% saturation of ammonium sulfate was shown to inherit the highest activity of tRNA-methyltransferases. In rabbit liver cytosol, the bulk of the tRNA-methyltransferase activity (approximately 50%) was found to be associated with high molecular weight complexes containing aminoacyl-tRNA-synthetases. The tRNA-methyltransferase activity was increased almost 1.4-fold both in the myocardium cytosol under total ischemia of isolated heart (30 min, 37 degrees C) and in liver cytosol under experimental myocardial infarction (EMI, occlusion of anterior coronary artery for 12 hours). Moreover, the labilization of high molecular weight complexes was observed: up to 80% of the tRNA-methyltransferase activity was localized in the fraction of lower molecular weight complexes and free enzyme fraction. In the total set of eight methylated nucleotides (products of submethylated tRNA methylation by liver enzymes), the decreased m1A content and the increased m7G and m1G contents were observed at EMI. It was assumed that the observed changes in the state of aggregation of tRNA-methyltransferases, in particular, their dissociation from the high molecular weight amino-acyl-tRNA-synthetase complexes are prerequisites for the suppression of protein biosynthesis under ischemic conditions.     

Physicochemical and biological properties of glucagon-like polypeptides from porcine colon.

Polypeptide material displaying glucagon-like immunoreactivity was isolated from porcine colon using immunoaffinity chromatography. The immunoreactive material was tightly bound to high molecular weight proteins but was dissociated by 0.1% w/v sodium dodecyl sulphate solution into immunoreactive components of approximate molecular weights 12,000,8000,5000 and 3000. These components reacted at least 50 times more strongly with antibodies specific for the N-terminal region of glucagon than with antibodies specific for the C-terminal region of glucagon. While the 8000 and 3000 dalton fractions were homogeneous, the 12,000 and 5000 dalton fractions were resolved into multiple bands by isoelectric focusing. The 12,000 dalton fraction was devoid of glycogenolytic and lipolytic activity, was not insulin releasing and showed no ability to bind to receptor sites specific for glucagon on hepatic plasma membranes and to active hepatic adenylate cyclase. The 8000 and 5000 dalton components showed weak lipolytic activity. The possible significance of colonic glucagon-like immunoreactivity relative to pancreatic glucagon and immunoreactivity from other tissues is discussed.     

The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin.

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).     

Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.     

Placental protein 5 is related to blood coagulation and fibrinolytic systems

Previous studies have shown that placental protein 5 (PP5) forms complexes with heparin. In order to further elucidate the biological role of PP5 we studied the effect of plasmin and thrombin on the immunoreactivity of PP5, and the possible functional antiplasmin and antithrombin effects of purified PP5. Varying concentrations of plasmin and thrombin were added to pregnancy plasma, and the PP5 levels, measured by radioimmunoassay, were found to be elevated by 558% (plasmin and 48–87% (thrombin). Incubation of radiolabeled PP5 with plasmin resulted in the formation of radioactive fragments with smaller molecular weights. Functional studies using a chromogenic substrate confirmed that purified PP5 has an anti-plasmin activity. An average increase of 15% was observed in the antiplasmin activity when 200 ng purified PP5 was added to 150 μl of pregnancy serum. Thus, there are certain similarities between PP5 and antithrombin III. Both form complexes with heparin and have antiplasmin properties, and both were found to be heat labile. But, functional studies utilizing a chromogenic substrate failed to demonstrate any antithrombin III-like activity in the purified PP5 preparation that had antiplasmin activity. Our results show that the function of PP5 is related to the blood coagulation and fibrinolytic systems, at least through its inhibitory action on plasmin.     

Inhibitor of trypsin and chymotrypsin in cultures of HeLa cells.

An inhibitor of trypsin and chymotrypsin with apparent molecular weight of 68 000 and a mobility similar to alpha1-globulin on polyacrylamide gel electrophoresis, was isolated from serum-free supernatant preparations from HeLa cells. Immunoelectrophoresis assays indicated that the inhibitor differed serologically from known inhibitors of serine proteinases in plasma and urine but shared antigenic determinants with an unidentified protein in these body fluids and with an inhibitor recently isolated from cultures of lung.     

High molecular weight complexes of folic acid in mammalian tissues

Twenty-four hours after injection of tritiated folic acid into normal rats, a large amount of labeled folates were found to be associated with the high molecular weight fraction of liver, kidney and intestine. The bound folate was associated with three fractions in the liver cell supernatant having approximate molecular weights of ? 350,000, 150,000 and 25,000 daltons, respectively. A fourth fraction which had an approximate molecular weight of 90,000 daltons was isolated from the liver nuclear fractions. The bound folates associated with these fractions were almost exclusively polyglutamate forms.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

Isolation of human complex-forming glycoprotein, heterogeneous in charge (protein HC), and its IgA complex from plasma. Physiochemical and immunochemical properties, normal plasma concentration

Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two major protein HC components in blood plasma. The plasma concentrations of the low and high molecular weight protein HC components were measured by this method in 13 healthy Caucasians. The results for the low molecular weight protein HC were: mean, 20.3 mg/liter, S.D., 3.2 mg/liter, range, 13.6-26.0 mg/liter; and for the protein HC X IgA complex: mean, 293 mg/liter, S.D., 176 mg/liter, range, 36-620 mg/liter.     

Inhibition of lipid transfer by a unique high density lipoprotein subclass containing an inhibitor protein

We have isolated from human plasma a unique subclass of the high density lipoproteins (HDL) which contains a potent lipid transfer inhibitor protein (LTIP) that inhibited cholesteryl ester, triglyceride, and phospholipid transfer mediated by the lipid transfer protein, LTP-I, and phospholipid transfer mediated by the phospholipid transfer protein, LTP-II. This HDL subclass not only inhibited cholesteryl ester transfer from HDL to LDL or VLDL, but also inhibited cholesteryl ester transfer from HDL to HDL. The inhibitor protein was isolated by sequential chromatography of human whole plasma on dextran sulfate-cellulose, phenyl-Sepharose, and chromatofocusing chromatography. Isolated LTIP had the following characteristics: an apparent molecular weight of 29,000 +/- 1,000, (n = 10) by sodium dodecyl sulfate gel electrophoresis, and an isoelectric point of 4.6 as determined by chromatofocusing. LTIP remained functional following delipidation with organic solvents. Antibody to LTIP was produced, and an immunoaffinity column of the anti-LTIP was prepared. Passage of human, rat, or pig whole plasma over the anti-LTIP column enhanced cholesteryl ester transfer activity in human (17%), pig (200%), and rat plasma (125%). The HDL subclass containing LTIP was isolated from whole human HDL (d 1.063-1.21 g/ml) by immunoaffinity chromatography. The isolated LTIP-HDL complex was shown to: i) contain about 60% protein and 40% lipid, ii) have alpha and pre-beta electrophoretic mobility, iii) have particle size distribution somewhat smaller than whole HDL, about 100,000 daltons, as determined by gradient gel electrophoresis, and iv) contain only a small amount of apoA-I (less than 5%) and a trace amount of apoA-II. Assay of ultracentrifugally obtained lipoprotein fractions revealed that approximately 85% of the total functional LTIP activity was in the d 1.063-1.21 g/ml HDL fraction. Furthermore, immunoblot analysis of whole plasma by nondenaturing gradient gel electrophoresis revealed that LTIP was found predominantly in particles in the size range of HDL. This unique HDL subclass may play an important role in the regulation of plasma lipid transfer and metabolism.