The presence of two GDP-L-fucose: glycoproteine fucosyltransferases in human serum

Two soluble fucosyltransferases have been demonstrated in human serum. One enzyme transfers l-fucose from GDP-l-fucose to the terminal galactose residues of lactose, N-acetyllactosamine, and sialidase-treated α₁-acid glycoprotein, to form the blood group H determinant, α-l-fucosyl-(1 → 2)-β-d-galactosyl-R. The second enzyme transfers fucose to the terminal N-acetylglucosamine residue of sialidase-, β-galactosidase-treated α₁-acid glycoprotein. Serum from a donor with the rare “Bombay” O_h blood group (genotype hh) cannot transfer fucose to terminal galactose residues but has normal levels of the enzyme acting on sialidase-, β-galactosidase-treated α₁-acid glycoprotein. This observation, as well as mixed substrate experiments, demonstrate that the two fucosyltransferase activities are due to two separate enzymes. The GDP-l-fucose:galactoside fucosyltransferase has a pH optimum of 5.5 and the following K_m values: lactose, 31 mm; N-acetyllactosamine, 7.5 mm; sialidase-treated α₁-acid glycoprotein, 6.4 mm. The GDP-l-fucose: N-acetylglucosaminide fucosyltransferase has a pH optimum of 5.0 and a K_m for sialidase-, β-galactosidase-treated α₁-acid glycoprotein of 1.2 mm. The serum GDP-l-fucose: N-acetylglucosaminide fucosyltransferase is distinct from the blood group Lewis-dependent enzyme in milk since the serum enzyme is present in serum from Le (a-b-)donors and since the Le-dependent fucosyltransferase could not be demonstrated in serum from donors carrying the Le gene.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Late spermatocytes from immature rat testis isolation,electron microscopy,lectin agglutinability and capacity for glycoprotein and sulfogalactoglycerolipid biosynthesis

A method is described for preparing late spermatocytes form immature rat testes which yields about 2 · 10⁵ cells per testis a purity of 70–80% and a viability of over 90%. The spermatocytes are highly agglutinable by both concanavalin A and wheat germ agglutinin but no major difference in lectin-mediated agglutinability was observed between late spermatocytes, early spermatids and spermatozoa. Isolated spermatocytes were capable of incorporating [¹⁴C]glucosmine into glycoprotein at a linear rate for about 50 min at 30°C and contained a glycoprotein N-acetylglucosaminyltransferase (8.6 nmol/mg protein per h) and a glycoprotein fucosyltransferase (4.5 nmol/mg protein per h) previously described in partially purified adult mouse testicular germinal cells. A Golgi-rich fraction was prepared from isolated spermatocytes wchich was enriched 15-fold in the N-acetylglucosaminyltransferase, 19-fold in the fucosyltransferase and 3-fold in a galactosyltransferase. These studies showed that late spermatocytes were higly active in glycoprotein synthesis. Studies on the incorporation of [³⁵S]sulfate into sulfogalactoglycerolipid indicated that late spermatocytes were not the primary site of synthesis of this lipid although late spermatocytes were shown to be highly enriched in sulfogalactoglycerolipid (5 times the level in whole rat testis). Further, [³⁵S]sulfogalactoglycerolipid took 5 weeks to migrate from its site of synthesis to the epididymis. These studies suggest that sulfogalactoglycerolipid is sulfated at a spermatocyte cell stage prior to the late (pachytene and diplotene) seprmatocyte stage.     

Properties and regional distribution of cerebral CMP-N-acetylneuraminic acid: Glycoprotein sialyltransferase

Glycoprotein sialyltransferase was studied in the rat brain and in the frontal grey cortex and corpus callosum of the calf brain. Activities were measured with endogenous acceptors as well as with desialized α₁-acid glycoprotein as an exogenous acceptor. The enzyme was characterized by means of its pH optimum, K_m values and requirements for detergent and cations. The properties of the rat and calf brain enzymes appeared to be very similar. Substrate specificity studies indicate that more than one glycoprotein sialyltransferase reaction may occur in brain. The regional distribution of the enzyme in the calf brain was rather uniform. From this it was concluded that glycoprotein sialyltransferase, at least for the greater part, is localized in membranes other than those of the synaptic complexes, and occurs in both neurons and glia cells. The regional distribution of the amounts of endogenous glycoprotein acceptor sites, which could be calculated from the sialyltransferase activities, showed a striking correlation with that of the protein-bound sialic acid, but not with the sialyltransferase activity. The role of these endogenous glycoprotein acceptors in cerebral sialoglycoprotein biosynthesis is discussed.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Characterization of serum, liver, and intestinal sialyltransferases from rats treated with colchicine

A modified high pressure liquid chromatographic method using lactose (Gal beta 1----4Glc) as an exogenous acceptor has been used to characterize the sialyltransferases known to increase in the serum of colchicine-treated rats. The results show a 10-fold increase of Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase (alpha 2----6 ST), whereas the Gal beta 1----3GlcNAc alpha 2----3 sialyltransferase showed only 1.6-fold increase in the serum after 17 h of colchicine treatment. The sialyltransferase activity in serum using exogenous desialylated, alpha 1-acid glycoprotein as acceptor also showed an eightfold increase. In liver homogenate and Golgi membrane, the sialyltransferase activity when assayed with desialylated alpha 1-acid glycoprotein as acceptor showed a slight decrease after 4 h, but returned to normal level after 17 h. A similar trend was seen when the two transferases were assayed with lactose as acceptor. The antiserum to rat alpha 2----6 ST inhibited the sialyltransferase activity in serum, liver, and jejunal incubation medium. Jejunal sections from rats treated with colchicine for 4 h in presence of heated serum showed a decrease of sialyltransferase, with consequent increase of the alpha 2----6 ST enzyme activity in the medium. This result suggests that intestinal tissue could be a source of increased serum enzyme activity in colchicine treatment.     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

Intracellular precursor forms of transferrin, α1-acid glycoprotein,and α1-antitrypsin in human liver

Ion-exchange chromatography of dialyzed human plasma and of buffer extracts of acetone-dried powder from human liver was used to analyze 13 different plasma proteins which are synthesized in the liver. Specific intracellular forms which differ from the plasma forms were found for transferrin, α₁-acid glycoprotein, α₁-antitrypsin, and albumin. The intracellular forms were labeled earlier than the plasma forms, when liver slices were incubated with radioactive leucine, suggesting that they are precursor forms of the proteins in the bloodstream. The liver form of transferrin was found to have the same molecular weight and N-terminus as the plasma form, but it differed from the plasma form by the absence of sialic acid. For α₁-acid glycoprotein two different liver forms were observed, both of which had lower molecular weights than the plasma form. One of these liver forms was analyzed further. Its polypeptide chain was found to have a blocked N-terminus, as does the plasma form. However, in contrast to the plasma form, it did not contain sialic acid. Its content of N-acetyl glucosamine was about one-third and the content of neutral hexoses about two-thirds of that found in the plasma form. Circular dichroism spectra were similar for liver and plasma forms and indicated a predominant β structure with very little α-helix content for both.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

The amino acid sequence of toxin V from Anemonia sulcata

Preparations of the β-galactoside-binding lectin of bovine heart have been shown to stimulate $\text{in vitro}$ the sialylation of the oligosaccharide Ga1β1→4G1cNAc and asialo-α₁-acid glycoprotein by bovine colostrum β-D-galactoside α2→6 sialyltransferase. Kinetic data revealed that in the presence of lectin the K_m values for Ga1β1→4G1cNAc and CMP-NeuAc were reduced from 25.0 to 11.6 mM and from 0.42 to 0.19 mM respectively, but the K_m for asialo-α₁-acid glycoprotein and the V_max values for all three substrates were little affected. Stimulation by the lectin was partially inhibited by Fucα1→2Ga1β1→4G1cNAc. This, together with the effects of certain plant lectins, suggests that the stimulation of sialytransferase may be mediated through the carbohydrate-binding properties of the lectin.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat

In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction.     

Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.     

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

The acute phase response to inflammation: the role of monokines in changes in liver glycoproteins and enzymes of glycoprotein metabolism

The role of monocyte derived factors in the acute phase response to inflammation is discussed. The kinetics of response of alpha 1-acid glycoprotein, sialyltransferase and albumin to a rat monokine preparation is described. There was an increase in synthesis of alpha 1-acid glycoprotein and sialyltransferase and a decrease in albumin synthesis following administration. However, the kinetics of response of sialyltransferase to the monokine was much slower than was found for the other two proteins. The possibility that sialyltransferase responds to a different monokine compared to the other acute phase proteins is discussed.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.     

Influence of sodium bis(oxalato)oxovanadate(IV) on phospholipids in liver Golgi fractions from control and streptozotocin-diabetic rats

(1) Both vanadyl oxalate and streptozotocin (STZ) caused in comparison with untreated control statistically significant increase (P<0.001 and P<0.02) of PLs (μmoles of P_i per mg of protein) in rat liver Golgi-rich membrane fraction. (2) The diabetic, vanadium treated rats (D+V) showed lower than control-treated (C+V) content of PLs in these fractions. (3) Three experimental groups of rats: control-treated (P<0.01), diabetic treated with vanadium (P<0.05) and untreated diabetic (P<0.02), had a higher percentage of PI (phosphatidylinositol) in comparison with untreated-control animals.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

The effect of α1-acid glycoprotein (orosomucoid) on triglyceride metabolism in the nephrotic syndrome

We have identified α₁-acid glycoprotein as a new co-factor in the lipoprotein lipase reaction. We isolated an active form of the compound from nephrotic urine that is effective both $\text{in vitro}$ and $\text{in vivo}$. α₁-acid glycoprotein increased lipolysis 100% in the presence of C-II apolipoprotein in a lipoprotein lipase assay system. Rats with induced nephrotic syndrome showed a decrease in triglyceride clearance. $\text{T}\text{1}\text{2}$ was increased from 14 min to 43 min. The injection of α₁-acid glycoprotein restored the lipid clearance to normal. These findings suggest that elevated plasma triglycerides in human nephrotic patients is the direct result of excessive loss of α₁-acid glycoprotein from plasma into urine. We propose that replacement therapy may be possible.     

A glycopeptide resistant to exoglycosidases

The carboxyl group of the terminal N-acetylneuraminic acid residue of the glycopeptide, prepared from α₁-acid glycoprotein by protease digestion, was esterified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then reduced with sodium borohydride. The reduced glycopeptide, thus prepared, containing the reduced N-acetylneuraminic acid, was resistant to hydrolysis by neuraminidase, and consequently to other exoglycosidases. The penultimate β-d-galactosyl residue of the oligosaccharide chain of the reduced glycopeptide was hydrolyzed by β-d-galactosidase only after the removal of the terminal, reduced, sialic acid by mild hydrolysis with acid. The reduced glycopeptide should be a useful substrate for the assay of endoglycosidases in the presence of exoenzymes. It should also find use as a carbon source in the growth of endoglycosidase-elaborating bacteria.