Structural glycoproteins from rabbit aortic media.

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.     

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.     

Separation of collagenase and a metal-dependent endopeptidase of rat uterus that hydrolyzes a heptapeptide related to collagen.

1. The synthetic peptide, 2,4-dinitrophenyl-L-Pro-L-Leu-Gly-L-Ile-L-Ala-Gly-L-Arg-amide (DNP-peptide) was tested as a potential substrate for uterine collagenase. Rat uteri were homogenized and the insoluble fraction was extracted at 60 degrees C to obtain collagenase. The extracts were chromatographed on Sephadex G-150 to yield two peaks of DNP-peptide hydrolyzing activity. Peak I was completely inhibited by EDTA and had a molecular weight greater than 100 000. Peak II was inhibited about 90% by EDTA and had an apparent molecular weight of about 70 000. 2. Peak II coincided closely, but not exactly, with the peak of collagenase activity. It differed from collagenase in heat stability, binding properties on CM-Sephadex and failure to display latency. 3. Peak II represents a new endopeptidase activity. It has a pH optimum of 7 and it cleaves the DNP-peptide at the Gly-Ile and, possibly, the Leu-Gly bond. 4. The DNP-peptide is not a satisfactory substrate for the assay of impure collagenase preparations nor does it inhibit the action of collagenase on collagen substrate when added in 30-fold molar excess.     

Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.     

Collagenase enzymes from Clostridium: characterization of individual enzymes.

Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.     

Guinea pig pancreatic acini prepared with purified collagenase

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca²⁺. Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with <0.14 units of protease per unit of collagenase produced highly responsive acini; collagenase with >0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.     

Glycolipid-binding proteins

Proteins which bind glycolipids with high specificity are tentatively divided into two groups. One group consists of activator proteins involved in the catabolism of glycolipids by acid lysosomal hydrolases. Two activator proteins, GM2-activator and sphingolipid activator protein-1, are critically appraised on their glycolipid-binding properties and on their activity to facilitate the transfer of glycolipids. These proteins are glycoproteins localized in the lysosomes. Their molecular weights are in a range of 21 000-27 000, and isoelectric points are 4-5. Glycolipid transfer protein (GLTP) is included in the other group. GLTP purified from pig brain has a molecular weight of about 20 000 and an isoelectric point of 8.3. GLTP facilitates the transfer of various glycosphingolipids and glyceroglycolipids between membranes. The protein does not facilitate the transfer of phospholipids or cholesterol. GLTP binds galactosylceramide. The galactosylceramide-GLTP complex participates in the transfer reaction as the intermediate. Each protein in both groups binds glycolipids with a characteristic specificity to the sugar moiety. A stoichiometry of 1 mol of lipid per mol of protein has been found in all three proteins. Proteins in both groups seem to have a hydrophobic region on their surface, since all three proteins have been efficiently purified by hydrophobic chromatography.     

Properties of pig synovial collagenase.

1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.     

Further evidence for non-pancreatic insulin immunoreactivity in guinea pig brain

The existence of large amounts of insulin in rat brain and of a porcine- or rat-like insulin in guinea pig brain have been disputed on the basis of differing results from standard (Method I) and hydrophobic adsorption techniques (Method II) for concentrating insulin from acid ethanol extracts. To try to resolve these differences, acid ethanol extracts of rat and guinea pig brains were divided into equal aliquots and concentrated for insulin radioimmunoassay (RIA) by both techniques. The RIA used guinea pig anti-porcine insulin serum, with 50% B0 for purified pancreatic porcine, rat and guinea pig insulin standards being 1.35, 2.38 and greater than 1,000 ng/ml, respectively. Oral glucose (4 g/kg) produced plasma glucose of 377 mg/dl in a guinea pig by 20 min but was not associated with any porcine- or rat-like immunoreactive insulin. Dilutions of guinea pig and rat brain extracts had parallel cross-reactivity with insulin standard curves. Insulin contents of rat brain (uncorrected for recovery) against porcine and rat insulin standards, respectively, were 1.33 and 1.93 ng/g (Method I) and 5.93 and 11.67 ng/g (Method II). Rat plasma was 0.85 and 1.42 ng/ml, respectively. Guinea pig contained 1.35 and 1.89 ng/g (uncorrected), respectively (Method I), and 2.99 and 5.62 ng/g, respectively (Method II). Guinea pig plasma was below the sensitivity of the RIA (less than 0.15 ng/ml). These results suggest that a porcine- or rat-like insulin may exist in guinea pig brain.     

Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig

Two different small proteins that cross-react with the antiserum against bovine caltrin (calcium transport inhibitor) have been purified from the seminal vesicle contents of the guinea pig. The primary structure and some molecular characteristics of the pure proteins are reported. The two proteins interact with concanavalin A indicating the presence of carbohydrates in their molecules. Chemical deglycosylation with trifluoromethanesulfonic acid, after reduction and carboxymethylation, results in complete loss of affinity for the lectin. Removal of sugar components from the structure destroys the ability of caltrin-like proteins to react with antibodies to bovine caltrin. The protein moving faster on polyacrylamide gel electrophoresis is designated guinea pig caltrin I, the other is II. They contain 45 and 55 amino acids, and the molecular weights of the peptide portions are 5082 and 6255, respectively. Although they have entirely different amino acid sequences, they share some common features: recognition by rabbit antibodies to bovine caltrin, the predominance of basic residues and the presence of 3 cysteine residues in fraction I and 8 in fraction II. The proteins have pI values of 9.5 and 10.2, respectively, which are consistent with the amino acid composition. The two pure fractions are approximately equally effective, on a weight basis, as inhibitors of 45Ca2+ uptake by guinea pig spermatozoa. The data presented reinforce the hypothesis that caltrin-like proteins are responsible for the previously reported (Coronel, C.E., San Agustin, J., and Lardy, H.A. (1988) Biol. Reprod. 38, 713-722), calcium-transport inhibitor activity detected in reproductive tract fluid from adult male guinea pigs.     

Molecular properties of multiple forms of acid phosphatase from horse liver.

1. Horse liver acid phosphatase was separated into two partially purified fractions differing in molecular weight (enzyme I about 100 00, enzyme II about 25 000). 2. Enzyme I was separated into several subfractions by DEAE-cellulose chromatography and isoelectric focusing. 3. Molecular weight, sedimentation coefficient and effective molecular radii were determined for acid phosphatases I and II by gel filtration and density-gradient centrifugation.     

Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans.

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.     

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Epidermal arachidonate lipoxygenase

Guinea pig skin was found to display a high lipoxygenase activity, evidenced by the formation of a hydroxyeicosatetraenoic acid (HETE) from exogenous [14C]arachidonic acid. The lipoxygenase activity was localized to the epidermal layer of the skin, was completely inhibited by eicosatetraynoic acid (ETYA) and slightly enhanced by indomethacin. Susceptibility to inactivation by sulfhydryl-directed reagents indicated that an essential sulfhydryl is present in a hydrophobic region of the molecule. The enzyme exhibited a broad pH activity optimum and a Km of 2.48 . 10(-5) M. The cytosolic enzyme has been partly purified by ammonium sulfate precipitation and two steps of of column chromatography and exhibited an apparent high molecular weight. The lipoxygenase and hydroperoxidase activities were resolvable from one another. The physiological and pathophysiological roles of the enzyme remain to be elucidated.     

Characterization of class II histocompatibility antigens in pigs

The D region of the SLA complex in the pig has been studied by immunochemical and sequential immunoprecipitation techniques as well as the redistribution of membranous antigens (capping). The molecules identified by the anti-la sera were solubilized by NP 40, purified on lectin and precipitated. Polyacrylamide gel electrophoresis under dissociating conditions shows that these molecules are made up of two chains whose molecular weights are 32 000 and 26 000 daltons respectively. Sequential immunoprecipitation and capping experiments indicate that two distinct types of la molecules exist. At least a part of the nylon-wood-adherent lymphocyte population expresses both types of molecules.     

Isolation of soluble proteins capable of stimulating aerobic plasmalogen biosynthesis

The terminal step during aerobic plasmalogen biosynthesis is catalyzed by a microsomal desaturase system which converts 1-O-alkyl-2-acyl-sn-glycerophosphoethanolamine to 1-O-alk-1'-enyl-2-acyl-sn-glycerophosphoethanolamine (ethanolamine plasmalogen). The reaction depends on oxygen and NAD(P)H and is stimulated 3-10-fold by soluble activating factors contained in the 100 000 X g supernatant. Two stimulating proteins have been isolated from pig kidney; the partially purified proteins have identical molecular weights (27 000) but differ in their respective isoelectric points (protein I, 5.1 and protein II, 4.9). Both proteins behave identically in the biochemical studies conducted. Exogenous substrate binds to the stimulating proteins; the transfer of ethanolamine, but not of choline phospholipids, from liposomes to microsomes is enhanced by the stimulating proteins. They stimulate plasmalogen synthesis from either exogenous or endogenous substrate (synthesized from alkylglycerophosphoethanolamine by microsomal transacylases). The stimulating proteins have no enzymatic activity themselves; it is suggested that they affect events within the membrane and function as specific mediators between the membrane-bound enzyme system and the lipophilic substrate.