E.H. Eylar

Cellular membranes: The isolation and characterization of the plasma and smooth membranes of hela cells

Abstract A method is described for preparation of membrane fractions from the 4000 g supernatant fluid, obtained from a Dounce homogenate of HeLa cells, using a discontinuous sucrose gradient (20–45% sucrose). Two fractions were obtained, the plasma membranes and the smooth internal membranes that appeared by electron microscopy to be free of nuclei, mitochondria, lysosomes, microsomes, ribosomes, dense bodies, or other recognizable cellular elements. The integrity of the plasma membranes were preserved as shown by electron microscopic examination; ruptured cellular envelopes were seen by phase microscopy. The degree of purity of the plasma membranes was shown by high increase in specific activity of the 5′ nucleotidase over the cellular homogenate of 120 fold. An ATPase, alkaline phosphatase, and phosphodiesterase were also found in the plasma membranes, giving a 30-fold, 21-fold, and 6-fold purification respectively. The 5′ nucleotidase, strongly bound to the plasma membranes, was located exclusively in these membranes. The yield of plasma membranes based on recovery of this enzyme was 40–50%. The absence of mitochondria, microsomes, and smooth membranes from the plasma membrane fraction was revealed by the failure to detect succinic dehydrogenase, esterase, and UDPase, respectively. The plasma membranes were characterized by a high molar ratio of cholesterol/phospholipid (1.05) which is similar to that found in myelin, and erythrocyte stroma. This ratio in the smooth membranes (0.06) illustrates the large difference in composition between these membranes and the plasma membranes. In contrast to the plasma membranes, found as a pellet below the 45% sucrose, the smooth internal membranes were found above the 25% sucrose level. They contained 61% lipid; the plasma membranes had only 29% lipid. Neither fraction contained appreciable amounts of RNA, but both had cholesterol, hexosamine, and sialic acid. The smooth internal membranes had the highest content and specific activity of UDPase, a Golgi enzyme. but the 5′ nucleotidase and ATPase, the plasma membrane enzymes, and succinic dehydrogenase, a mitochondrial enzyme, were not detected. Other enzymes found in the smooth membranes included alkaline phosphatase, esterase, and phosphodiesterase. The amino acid composition of the protein contained in the two membrane fractions was similar; the protein of the smooth internal membranes was characterized by 151 basic amino acid residues per 1000 residues and 221 acidic residues while the plasma membrane protein contained 193 basic and 188 acidic residues per 1000 amino acid residues. The glutamic acid content exceeded the other amino acids in both fractions.

Allergic encephalomyelitis: the physico-chemical properties of the basic protein encephalitogen from bovine spinal cord

Abstract A protein encephalitogen, isolated in a homogeneous state from bovine spinal cord, was found to have unusual properties. It was highly basic, containing 38 moles/ mole of basic amino acids (arginine, lysine, histidine) and only 7 moles/mole of acidic amino acids (glutamic and aspartic acid); the calculated isoionic point exceeded 12. Only one tryptophan (determined by three independent methods) and two methionine residues were present; no carbohydrate or lipid was associated with the isolated protein. Although similar to certain histone fractions, it differed from these proteins in its much higher content of histidine, glycine, and serine, and its lower content of alanine. The N-terminal position is blocked. The molecular weight determined by sedimentation equilibrium techniques was 16,400 daltons; by sedimentation-viscosity, 16,200; and by UV absorption studies based on the tyrosine-tryptophan content, 16,200. Estimates of molecular weight based on the amino acid analysis gave values of 15,500–18,200 daltons. An intrinsic viscosity of 9.27 ml/g and viscosity increment of 12.9 were found. From these data an axial ratio (a/b) of approximately 10:1 (assuming no hydration and a prolate ellipsoid) was calculated, revealing that the molecule is highly unfolded and approximates a rod-like shape. This property accounts for the unexpected behavior of this basic protein on gel nitration which has led to erroneously high molecular weight values. A small degree of aggregation was observed as indicated by the high molecular weight estimate of 22,300 found near the cell bottom using a long-column sedimentation equilibrium technique. No evidence of aggregation was observed at pH 2.6, 7.0, or 9.5 during sedimentation velocity studies. An S 0 20 , w value of 1.72S was found at pH 2.6, ionic strength 0.25. Considerable variation of the S 20, w was found with protein concentration, but not with pH. The highly extended conformation of the Al molecule, found from viscosity studies, is compatible with its resistance to denaturation; no loss in encephalitogenic activity was found following heating at 100 ° for 1 hr, treatment with 8 m urea for 8 hr, or incubation at pH 10.0 for 8 hr. Moreover, these procedures do not diminish interaction with antibody measured by the Ouchterlony and passive hemagglutination inhibition tests. Polyacrylamide gel electrophoresis also revealed no drastic changes in conformation arising from these procedures. It was concluded from these data that hydrophobic and hydrogen bonding play a minor role in the overall conformation of the A1 protein molecule. This property is of interest in considering the role of this protein as a membrane component.

Experimental Allergic Encephalomyelitis: Synthesis of Disease-Inducing Site of the Basic Protein

A highly encephalitogenic peptide whose structure resembles the sequence of amino acids surrounding the single tryptophan residue in the encephalitogenic A1 protein from bovine myelin was synthesized. This peptide is similar in the sequence to peptic peptide E and tryptic T27, derived directly from the A1 protein, and is as active on a molar basis as the A1 protein. The major disease-inducing site of the A1 protein resides in a linear sequence of nine amino acids: H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. This region of the A1 protein is apparently the major encephalitogenic determinant since specific modification of the tryptophan residue in the A1 protein with 2-hydroxy-5-nitrobenzyl bromide destroyed its encephalitogenic activity.

The biosynthesis of mannose-containing glycoproteins: A possible lipid intermediate

Abstract Particulate fractions from liver, oviduct, and myeloma tumor incorporated mannose- 14 C from GDP-mannose- 14 C into glycoprotein and lipid. Smooth membranes from rabbit liver were purified 8-fold with respect to incorporation of mannose- 14 C into endogenous lipid acceptor; the mannolipid appeared similar in many respects to the mannosyl-1-phosphoryl-polyisoprenol compound found in bacteria. Incorporation into lipid was 7 times greater than into protein when incubated in the presence of 6 × 10 −3 M MnCl 2 , 2.5 × 10 −3 M EDTA, and 0.15% Zonyl A. The role of lipid as a possible intermediate in the biosynthesis of secreted, soluble glycoproteins is discussed; a minimum of two mannosyl transferases are required.

Glycoprotein biosynthesis: The localization of polypeptidyl: N-acetylgalactosaminyl, collagen: Glucosyl, and glycoprotein: Galactosyl transferases in HeLa cell membrane fractions

Abstract Membranes from HeLa cells were purified by centrifugation in a discontinuous sucrose gradient. The distribution and properties of three glycoprotein:glycosyl transferases within these membranes was determined. A multienzyme group of glycosyl transferases, which is involved in the assembly of the carbohydrate units of membrane grycoproteins, was found almost entirely in the fraction representing the smooth internal membranes. Two of the enzymes of this group include a polypeptidyl: N -acetylgalactosaminyl transferase that catalyzes the synthesis of a proteincarbohydrate linkage and a glycoprotein:galactosyl transferase that attaches galactose to N -acetylglucosamine residues during the assembly of the carbohydrate units of the glycoprotein. The N -acetylgalactosaminyl transferase, for example, was purified 45–50-fold, representing 50–60% of the total cellular activity, during the course of purification of the smooth internal membranes. Each of these enzymes is strongly bound to the membranous structure, but can be solubilized with the nonionic detergent, Triton X-100. In contrast to this multienzyme group of the smooth membrane fraction, a collagen:glucosyl transferase, responsible for attachment of the terminal glucose residue to the carbohydrate portion of the secreted protein, collagen, was located exclusively in the plasma membrane. The glucosyl transferase was purified over 140-fold, representing 55% of the total activity of the cell, during the course of purification of the plasma membranes. The specific location of this enzyme suggests it to be a useful marker for the plasma membrane. On the basis of these data, it was suggested that the attachment of carbohydrate to proteins (extracellular) may be a necessary prelude to their secretion. The collagen:glucosyl transferase also differed from the transferases of the smooth membranes in its binding relationship to the membranous structures; it expresses full activity in the membrane-bound state, whereas the transferases require solubilization with Triton X-100 for maximum activity. With regard to the mechanism of glycoprotein biosynthesis, this work supports the concept of a “one enzyme-one linkage” concept in which each monosaccharide is added individually by a highly specific transferase to the growing carbohydrate chain. The intracellular site for assembly of the carbohydrate units of membrane glycoproteins appear to be the smooth internal membranes rather than the microsomes.

Glycoprotein biosynthesis: Studies on the receptor specificity of the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands☆

Abstract The receptor requirements of the polypeptidyl:N-acetylgalactosaminyl (galNAc) transferase, purified from bovine submaxillary glands, was investigated. The transfer of galNAc-14C to the hydroxyamino acid residues of the polypeptide chain of submaxillary glycoprotein denuded of carbohydrate is a highly specific process. All other proteins and glycoproteins studied, intact and partially degraded, were nonfunctional in this reaction except for the submaxillary polypeptide. Receptors were obtained from desialized bovine submaxillary glycoprotein (BSM) by three procedures: (1) removal of the N-acetylhexosamine residues by epididymal hexosaminidase; (2) periodate treatment followed by reduction with NaBH4 and mild acid hydrolysis; and (3) directly by acid hydrolysis (0.2 n H2SO4, 2 hr 100 °). Up to 17–22% of the N-acetylhexosamine previously removed could be replaced by the galNAc-14C using the purified transferase. The best receptor material was obtained by removal of 50–70% of the N-acetylhexosamine by hexosaminidase or the periodate procedure. A linear relationship was found between the percentage of N-acetylhexosamine removed by hexosaminidase, up to approximately 50%, and the incorporation of galNAc-14C. When more than 70–75% of the N-acetylhexosamine had been removed, however, no further increase in receptor activity was found; upon removal of 75% or more of the N-acetylhexosamine, the receptor activity was significantly decreased. Materials such as intact BSM or desialized BSM, from which the N-acetylhexosamine had not been removed, were nonfunctional as receptors. The hydroxyl groups of sugar residues would not substitute for those of specific hydroxyamino acids of the submaxillary polypeptide chain. In this respect, N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid were all nonfunctional as receptor sites as demonstrated by the inactivity of bovine and porcine submaxillary glycoproteins, the α1-glycoprotein, and fetuin, each of which had been partially degraded with specific glycosidic enzymes. The specificity of the reaction for particular hydroxyamino acids of the BSM polypeptide receptor was inferred by the inactivity of the submaxillary glycoprotein after alkali-borohydride treatment. Twenty-five proteins, native and denatured, were tested for receptor activity with negative results. The only material 2 with receptor activity was found in the extract of the submaxillary glands and most likely represents endogenous BSM material at incomplete stages of synthesis. The receptors prepared from BSM exhibited both conformational and size requirements although they were unusually stable to some conditions known to denature proteins. In spite of treatment with 0.2 n H2SO4, 90 °, 2 hr, or with 65% ethanol, receptor activity was not affected. After the use of 8 m urea or heating at 100 °, 5 min, approximately 50% of the receptor activity still remained. The size requirements of the hydroxyamino acid-containing polypeptide chain was illustrated by the complete loss of receptor activity after Pronase digestion. After trypsin treatment, which yields larger peptides than Pronase, 35% of the receptor activity still remained. Small molecules, such as serine, threonine, triserine, trithreouine, and a pentapeptide containing both serine and threonine were also inactive. The results from this work emphasize the high specificity required of the gly cosyl transferases in general for synthesis of the specific structures found in the oligosaccharide units of glycoproteins. The existence of the polypeptidyl: galNAc transferase is consistent with a proposed “one enzyme-one linkage” concept of glycoprotein biosynthesis.

Cellular membranes: The biosynthesis of glycoprotein and glycolipid in HeLa cell membranes☆

Abstract The biosynthesis of glycoprotein and glycolipid in HeLa cells was studied by following the distribution of glucosamine- 14 C, fucose- 14 C, and leucine- 3 H as a function of time. The HeLa cells were useful for studying the biosynthesis of membrane components; nearly all of the glycoprotein and glycolipid synthesized by these cells is membrane-bound or intracellular. The extracellular material constituted only 1–4%. Most of the glucosamine- 14 C and fucose- 14 C was incorporated into glycoprotein and glycolipid found in the smooth internal and plasma membranes; by contrast, the majority of the leucine- 3 H was found in the microsomal fraction and soluble protein. It was concluded that glucosamine is not attached to protein at the ribosomal or microsomal level. From chase experiments it appeared that the soluble and smooth membrane glycoprotein and glycolipid was first labeled; the labeled material then migrates progressively to the plasma membrane. A hypothesis was proposed concerning the events involved in membrane glycoprotein biosynthesis and the localization of these events within the cell. The smooth internal membranes were specified as the subcellular site for assembly of membrane glycoprotein and glycolipid. The incorporation of glycoprotein and glycolipid membrane subunits, which are eventually integrated into the substructure of the plasma membrane, was discussed.

Glycoprotein biosynthesis: The characterization of two glycoprotein: Fucosyl transferases in HeLa cells☆☆☆

Abstract Two glycoprotein: fucosyl transferases that transfer fucose from GDP-fucose onto glycoprotein receptors were found in a Triton X-100 extract of HeLa cells. One enzyme, designated the fetuin:fucosyl transferase, utilizes as a receptor fetuin from which sialic acid and galactose were removed, thus leaving an available terminal N -acetylglucosamine residue. This enzyme has an optimum pH of 6.0, temperature optimum of 30 °, and requires Mg 2+ . The other enzyme designated the PSM: fucosyl transferase, utilizes as a receptor PSM from which sialic acid and fucose were removed leaving an available terminal galactose residue. This enzyme has an optimum pH of 6.8, temperature of 37 °, and does not require metal ion activation. Both enzymes showed a high degree of specificity when tested with various glycoprotein receptors. It was concluded that one enzyme was involved in formation of a fucosyl- N -acetylglucosamine linkage, probably 1–4, while the other enzyme recognized only galactose with probable formation of the α- l -fucosyl-(1–2)- O - d -galactose linkage found in blood group substances and porcine submaxillary glycoprotein. The galactose residues of fetuin and α 1 -glycoprotein were relatively inactive as receptors. Approximately 1.1–2.4% of the theoretical receptor sites reacted with GDP-fucose after 24-hr incubation. Both enzymes required macromolecular receptors; several mono- and disaccharides were nonfunctional as receptors. Both enzymes were were found in the smooth internal membranes containing the multienzyme group of glycosyl transferases involved in biosynthesis of the carbohydrate units of membrane glycoproteins. The fucosyl transferases are strongly bound to these membranes; dissociation with the detergent Triton X-100 solubilizes the enzymes. The detergent-solubilized enzymes were purified 11-fold by gel filtration on Sephadex G-200 followed by centrifugation at 300,000 g for 4 hr. The role of the fucosyl enzymes in the mechanism of glycoprotein biosynthesis is discussed. Of special interest in this regard is the synthesis of “artificial” glycoproteins illustrated by the replacement of the β-galactosyl-(1–4)- n -acetylglucosamine linkage present in native fetuin by the fucosyl- N -acetylglucosamine linkage formed with the fetuin:fucosyl transferase.

Allergic encephalomyelitis: Preparation of the encephalitogenic basic protein from bovine brain☆

Abstract A homogeneous preparation of the encephalitogenic A1 protein was obtained from the bovine brain or spinal cord by cation-exchange chromatography on either Bio-Rex 70 or Cellex P. The preparations appeared homogeneous by polyacrylamide gel electrophoresis at pH 4.3 and 8.9 and migrated identical to the basic protein prepared from bovine spinal cord. These preparations were encephalitogenic at the levels of 0.1 μg or higher per guinea pig and combine with antibody produced against the bovine spinal cord basic protein as shown by the passive hemagglutination inhibition test. It was concluded that the A1 protein is identical, whether derived from brain or spinal cord, and that its properties are independent of the final method of purification following acid extraction from the defatted tissue.

Allergic encephalomyelitis: a comparison of the encephalitogenic A1 protein from human and bovine brain.

Abstract The chemical and biological properties of the human encephalitogenic basic protein was compared to that from bovine brain. Both proteins are highly basic and contain approximately 25 net positive charges at pH 6. They each contain one residue of tryptophan per mole but no cysteine. The electrophoretic migration in polyacrylamide gel at pH 4.3 and 8.0 was almost identical. The sedimentation coefficient was estimated to be 1.34 S, and their molecular weight 18,000–19,000 daltons with the human protein possibly larger. The optical rotatory dispersion studies suggested a random-coil structure; no α-helix or β-form was indicated. The encephalitogenic activity of the two proteins measured in the guinea pig was approximately the same. Their ability to combine with antibody prepared against the bovine protein was similar as determined by the hemagglutination-inhibition test. Both proteins were found to induce a significant delayed skin reaction in guinea pigs regardless of whether the human or bovine protein was used to produce sensitized cells. Thus, the physical, chemical, and biological properties of these two basic proteins are similar; the amino acid sequence is not identical, however, as revealed by the tryptic peptide maps.