Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

Histochemical analysis of sialic acids in the epididymis of the rat

 A variety of sialic acids contained in the rat epididymis were histochemically examined by means of lectin and pre-lectin methods by light microscopy. Epididymides from adult male Sprague-Dawley rats were fixed in Bouin’s fluid and routinely embedded in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidation–phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The present results revealed that principal cells in the initial segment and caput contain sialic acid linked to α2,6-galactose/N-acetylgalactosamine, whereas those in the corpus and cauda include the sialic acidα2,3-galactose sequence. Narrow and clear cells involve all the types of sialic acids examined. Basal and halo cells mainly contain sialic acidα2,3-galactose. 8- And/or 9-O-acetylated sialic acids were predominantly distributed in principal cells of the initial segment and proximal caput. These findings are taken to indicate that various sialic acids in the epididymis could participate in different physiological functions characteristic of the regions in this organ. Accepted: 10 November 1997     

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures

Summary Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Cullinget al. (1976), have been devised for the histochemical identificantion of side-chainO-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiffborohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chainvicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chainO-acylated sialic acids.     

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins

Summary A differential diagnostic scheme is described for the division of colonic epithelial glycoproteins into eleven histochemically distinct classes. The scheme depends upon the use of seven histochemical techniques which, collectively, permit the differential staining ofO-sulphate ester, sialic acid and its side chainO-acyl variants and vicinal diols located on carbohydrate residues other than sialic acids. Elements of the scheme also provide a general approach to the classification of epithelial glycoproteins in anatomic sites other than the colon.Application of the scheme permitted the classification of the epithelial glycoproteins in the mucosa 0.5–5.0 cm from human colonic tumours and provided direct confirmation of previous observations that changes from normal in the relative proportions of either side chainO-acylated sialic acids or sialic acids andO-sulphate esters can occur independently of one another.     

Genetic polymorphism of human urine deoxyribonuclease I

Summary A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes —DNASE1 1, 1–2, and 2 — and the other four rare phenotypes — DNASE1 1–3, 2–3, 2–4, and 3–4 — represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 ^* 1, ^* 2, ^* 3, and ^* 4. The frequencies of the DNASE1 ^* 1, DNASE1 ^* 2, DNASE1 ^* 3, and DNASE1 ^* 4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

Recognition of glycoconjugates byHelicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides,a second sialic acid-based specificity

Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way. The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins. The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12. Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids.Bacteria grown in F12 medium were metabolically labelled with³⁵S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus. There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides. In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level. The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis. Erythrocyte proteins as well as a range of reference glycoproteins did not bind, except band 3, which was weakly active. However, this activity was resistant to periodate oxidation.These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity. Abbreviations: PGCs, polyglycosylceramides; TLC, thin-layer chromatography; C, chloroform; M, methanol; EI/MS, electron impact ionization mass spectrometry, SDS PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin. The carbohydrate and glycosphingolipid nomenclatures are according to recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (Lipids (1977)12: 455–68;J Biol Chem (1982)257: 3347–51 andJ Biol Chem (1987)262: 13–18).This paper is dedicated to Professor S.-i. Hakomori and is paper no. 1 from our research onHelicobacter pylori.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. III. Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours

Summary Histochemical, chemical and histological studies were performed on 26 specimens of human colonic tumours and 62 specimens of mucosa taken at distances of 0.5–5.0 cm from the tumour. The tumour glycoproteins were divided almost equally between three anionic types, sulphomucin, sialomucin and mixed sialomucin and sulphomucin. All showed a reduction in staining for side chainO-acylated sialic acid. In 56% of the tumours, this was accompanied by loss of glycoprotein while, in 44%, abundant mucin was still present.Histochemical examination of the mucosal specimens indicated that in 24.2% the side chainO-acylated sialic acids did not differ from normal. In 41.9% there was a focal change and in 33.9% there was a generalized field reduction in the proportion of side chainO-acyl sialic acids. The latter were subdivided into moderate and severe. Chemical analyses correlated well with the histochemical classification of the mucosal specimens and showed that, on average, the classifications focal and severe field change were not due to sampling error. Forty-five per cent of the cases showed only focal change and 40% only field change. Mucosal specimens associated with 60% of the moderately differentiated tumours showed only focal change while those associated with 75% of well-differentiated tumours showed only field change. Abnormal patterns of staining for side chainO-acylated sialic acids (a) were largely independent of the distance from the tumour, (b) occurred in the presence of a normal pattern of staining for sialomucins and sulphomucins and (c) were associated with 61.4% of the specimens that showed no discernible evidence of histological abnormality. In contrast, only one specimen showed evidence of histological change without a corresponding change inO-acylated sialic acids. The data suggest that abnormal patterns of staining forO-acylated sialic acids may represent premalignant change but their precise significance and specificity requires further studies of non-neoplastic diseases of the colon.     

Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [¹⁴C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [¹⁴C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

Quantitative gas—liquid chromatography of neutral sugars in human serum glycoproteins : Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma

A precise and accurate gas—liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars -fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis times (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m × 2 mm I.D. column with 2.0% Silar-7 CP on Chromosorb W AW 80–100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50W-X8 (H⁺) ion-exchange resin in 0.5 N HCI at 100° for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of <86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC—mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.     

High-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil in cattle muscle tissue with on-line anion-exchange clean-up

A high-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil has been developed. The drug was extracted from cattle muscle tissue with 1% triethylamine in acetonitrile. The extract was evaporated to dryness and taken up in 0.1 M ammonium acetate—acetonitrile (50:50, v/v). The extract was then injected onto a polymeric anion-exchange precolumn. After clean-up with 0.1 M ammonium acetate—acetonitrile (50:50, v/v) for 5 min, the precolumn was eluted with 1% aqueous trifluoroacetic acid—acetonitrile (50:50, v/v) onto a PLRP-S polymer column and chromatographed with a mobile phase of 0.01 M phosphate pH 7—acetonitrile (80:20, v/v). Detection was by ultraviolet at 273 nm. Average recoveries at four levels from 0.005 to 1.000 mg kg⁻¹ were > 88%. The limit of determination was 0.005 mg kg⁻¹.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. I. A comparison between histologically normal colon, colonic tumours, ulcerative colitis and diverticular disease of the colon

Summary Chemical and histochemical methods were used to compare the epithelial glycoproteins from formalin-fixed surgical specimens of normal human large intestine, colonic tumours, ulcerative colitis and diverticular disease. All the epithelial glycoproteins contained fucose, galactose, glucosamine, galactosamine and, in addition, sialic acids both with and withoutO-acyl substituents in the side chain and/or at position C₄. The glycoproteins of the normal ascending and descending colons differed significantly with respect to the percentage of the sialic acids released following digestion of the de-O-acylated glycoprotein withVibrio cholera neuraminidase and to the molar fucose-sialic acid ratio. Statistical analysis of the chemical data showed that (a) compared to normal, the sialic acids of the tumour and ulcerative colitis glycoproteins from the descending colon were significantly less substituted in the side chain and at position C₄; (b) theO-acetyl substitution pattern of the sialic acids of the ulcerative colitis glycoproteins from the ascending colon and the quantitative composition of the carbohydrate prosthetic groups of the ulcerative colitis glycoproteins from both ascending and descending colons differed from normal; (c) it was not always possible to distinguish between the ulcerative colitis and tumour glycoproteins on the basis of theO-acetyl substitution pattern of their sialic acids; and (d), there were minor differences between normal glycoproteins and those from cases of diverticular disease.     

Chemical and histochemical studies of normal and diseased gastrointestinal tract. IV. A comparison of histochemical and chemical methods for the estimation of side chain O-acylated sialic acids

Summary Statistically significant correlations were obtained between a chemical assay for the proportion of colonic epithelial glycoprotein sialic acids with side chainO-acyl substituents and two histochemical methods, the PBT-KOH-PAS sequence (r _s=0.7485 forN=31,P=0.01, one-sided test) and the PAPT-KOH-Bh-PAS procedure (r _s=0.7024 forN=34). A positive correlation (r _s=0.8654 forN=30,P=0.01) was also obtained between the results of the two histochemical procedures. It is concluded that, on average, histochemical observations are a reliable semiquantitative comparative method for the estimation of side chainO-acetylated sialic acids.     

Biochemical examination ofRana pipiens epithelial mucus

Summary The epithelial mucus ofRana pipiens is shown to be highly negative by histochemical procedures, uptake of tagged extracellular markers, equilibrium dialysis, and QAE-Sephadex G-25 ion exchange chromatography. The mucus is found to contain 0.4% (dry weight) sulfate, 16% (dry weight) protein and 9% (dry weight) neutral sugars, whereas no sialic acid is detected. A mucin charge of –40 equivalents mole^–1 is calculated by equilibrium dialysis using a molecular mass of 100,000 Dalton. An independent determination of in situ charge density by radionuclide uptake onto the frog surface (1.55 meq l^–1) suggests that this estimate of mucin charge is correct. Based on analysis of size-exclusion chromatography fractions, it is suggested that mucus contains a sulfated glyco-protein (100,000 Dalton) that forms aggregates of about one million Dalton.     

Protein composition and structure of human placental microvillous membrane. External surface, sialic acid containing, extrinsic and intrinsic components.

The microvillous membrane of human placenta is in direct contact with maternal blood and thus plays a vital role in many essential functions of the placenta. As an initial step in understanding the membrane proteins, and their relationship to these functions and to the structure of the membrane, we have investigated an isolated membrane preparation. Ten major peptide bands and an approximately equal number of minor bands were seen with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Sialoglycoproteins were labeled with periodate (PA-³H) borohydride and external surface components with lactoperoxidase-[¹²⁵I] (LP-¹²⁵I). One principal (69 000 mol. wt) and several minor (100 000, 45 000, and 38–40 000 mol. wt) bands were labeled as Sialoglycoproteins and found to be exposed on the surface of the membrane. Approx. 50% of the membrane protein and all of the sialic acid was tightly bound to membrane lipid and resistant to extraction with dimethyl maleic anhydride (DMMA). Electron microscopy demonstrated extraction by DMMA of microfilaments presumptively identified as actin and other electron dense components from the villous core. The extracted supernate and the residual pellet differed markedly in protein composition. The supernatant contained bands of 180 000, 115 000, 85 000, 70–72 000, 45 000, and 38–40 000 mol. wt whereas the lipid pellet contained components of 200 000, 150 000, 100 000, 69 000, and 64 000 mol. wt. The lipid matrix with which these proteins were associated contained phosphatidyl choline and sphingomyelin and was similar in composition to other plasma membranes. Thus by using a variety of experimental approaches the proteins of the human placental microvillous membrane can be divided into groups based on their sialic acid content, exposure on the external surface, tightness of binding to the membrane lipid, and relation to membrane structure.