Tumour targeting: biological factors and formulation advances in injectable lipid nanoparticles.

Cancer chemotherapeutic agents are often administered systemically. Following systemic administration, numerous biological factors associated with the tumours influence the delivery of the drugs to the tumours. These factors have been extensively studied for the last 2 decades. The influence of these biological factors has brought about a drastic change in the design of drug delivery systems to solid tumours. This review discusses the various biological factors influencing drug delivery to tumours and the subsequent development of injectable delivery systems (i.e., lipid-based nanoparticles (SLNs)) for adequate delivery of drug to solid tumours.

A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality.

Formation of the dolichol oligosaccharide precursor is essential for the production of asparagine- (N-) linked oligosaccharides (N-glycans) in eukaryotic cells. The first step in precursor biosynthesis requires the enzyme UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT). Without GPT activity, subsequent steps necessary in constructing the oligosaccharide precursor cannot occur. Inhibition of this biosynthetic step using tunicamycin, a GlcNAc analog, produces a deficiency in N-glycosylation in cell lines and embryonic lethality during preimplantation development in vitro, suggesting that N-glycan formation is essential in early embryogenesis. In exploring structure-function relationships among N-glycans, and since tunicamycin has various reported biochemical activities; we have generated a germline deletion in the mouse GPT gene. GPT mutant embryos were analyzed and the phenotypes obtained were compared with previous studies using tunicamycin. We find that embryos homozygous for a deletion in the GPT gene complete preimplantation development and also implant in the uterine epithelium, but die shortly thereafter between days 4-5 postfertilization with cell degeneration apparent among both embryonic and extraembryonic cell types. Of cells derived from these early embryos, neither trophoblast nor embryonic endodermal lineages are able to survive in culture in vitro. These results indicate that GPT function is essential in early embryogenesis and suggest that N-glycosylation is needed for the viability of cells comprising the peri-implantation stage embryo.

Genomic organization and promoter activity of glucosidase I gene.

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.

Processing alpha-glucosidase I is an inverting glycosidase.

Alpha-glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the alpha-glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(alpha1-2)Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 was directly monitored by 1H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released beta-glucose concomitant with the formation of the Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.

Developmental and hormonal regulation of protein N-glycosylation in the mammary gland.

Glycosylation represents the most common conjugation of both membrane-bound and secreted proteins of animal cells. Among the different types of glycosylation, the N-linked attachment of sugars to the polypeptide backbone is by far the most abundant modification. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise assembly of Glc3Man9GlcNAc2P-P-Dol in the dolichol cycle, its transfer en bloc to the nascent polypeptide in the rough endoplasmic reticulum (RER), followed by excision of the glucosyl residues by processing-specific enzymes, glucosidase I and II, also resident in the endoplasmic reticulum. Additional posttranslational modifications of the carbohydrate moiety in the RER, Golgi, and trans-Golgi network, differ for individual glycoproteins for the completion of final products as high mannose, complex or hybrid glycoproteins en route to their final destinations in the secretory pathway. The enzyme GlcNAc-1-P transferase (GPT) catalyzes the first and committed step, i.e., the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P to form GlcNAc-P-P-Dol, in the assembly of the oligosaccharide precursor. Glucosidase I triggers the maturation phase by clipping the distal alpha 1,2-linked Glc residue on the incipient glycoprotein. The critical juxtaposition of the two enzymes in the multistep pathway makes them excellent candidates for the overall regulation of protein N-glycosylation. The highly elevated needs of glycosylation during lactation demand regulation of glycosylation in the gland over and above the levels in the quiescent, virgin and postlactating, regressed gland.

Structure-function relationships in glucosidase I: amino acids involved in binding the substrate to the enzyme.

As the enzyme that initiates the maturation phase of the oligosaccharide moiety of N-linked glycoproteins, glucosidase I controls the flux of carbohydrate during the biosynthesis of these proteins. In a previous study to elucidate the structure-function relationships, we reported the presence of a cysteine residue at or near the active site of the enzyme from the bovine mammary gland (Pukazhenthi,B.S., Muniappa,N. and Vijay,I.K., 1993, J. Biol. Chem., 268, 6445-6452). We have now extended this approach to identify the participation of an arginine and a tryptophan residue in the enzyme that may play an important role in binding the substrate. The data have been combined with the results of the previous study and the cDNA-derived sequence to propose a ERHLDLRCW motif in the active site of the enzyme in the rat mammary gland that is involved in binding the incipient glycoprotein substrate for processing.

Further characterization of negative regulatory element involved in the hormonal regulation of GlcNAc-1-P transferase gene in mouse mammary gland.

The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. Earlier, we demonstrated that a distal negative regulatory element in mouse GPT (mGPT) promoter plays an important role in developmental and hormonal control of mGPT gene expression in mammary gland (Ma J, Saito H, Oka T and Vijay IK (1996) J Biol Chem, in press). In this report, a tissue distribution of the repressor that binds the negative regulatory element was examined; a comparison of the negative regulatory element to other consensus sequences for known transcription factors is discussed.

Negative regulatory element involved in the hormonal regulation of GlcNAc-1-P transferase gene in mouse mammary gland.

The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. The involvement of cisacting elements in regulating the expression of the mouse GPT (mGPT) gene was investigated by transient transfections of various GPT promoter/luciferase (Luc) constructs into primary mouse mammary epithelial cells. A series of 5'-deletions of the GPT promoter identified a distal negative regulatory region (base pairs -1057 to -968) and deletion of this region results in enhanced hormonal induction (approximately 7-fold) with no effect on basal promoter activity. Electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from different developmental stages of mouse mammary gland demonstrated that the binding activity of the nuclear proteins to the distal negative regulatory region was predominant in virgin stage as compared with pregnant and lactating stages. EMSA performed with nuclear extracts from virgin explants showed that the binding activity was markedly decreased after cultivation with the combination of the three lactogenic hormones. DNase I footprinting analysis identified two pentamer direct repeat motifs, AGGAA and GAAAC, within the negative regulatory region. EMSA competition experiments showed that mutations within the direct repeats failed to compete for binding of the nuclear proteins to labeled wild type oligonucleotide. Transcription from the promoter containing the mutated direct repeats was increased greatly, consistent with the conclusion that these motifs functions in vivo to repress GPT gene expression. These data suggest the importance of the negative regulatory region in hormonal control of mGPT gene expression in mammary gland.

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

Developmental and hormonal regulation of UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferase in mouse mammary gland.

The developmental and hormonal regulation of UDP-GlcNA:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme which initiates the biosynthesis of asparagine-linked glycoproteins, was investigated in mouse mammary glands. An anti-peptide antibody raised against the carboxyl-terminal 11 amino acids of mouse GPT, immunoadsorbed GPT activity and recognized a protein of expected size (approximately 48 kDa) on Western blots. Mouse mammary glands at different stages of development were examined for GPT activity, immunoreactive protein, and GPT mRNA. All three parameters showed a similar trend, i.e. they were low in tissues from virgin and pregnant animals, increased steadily during lactation, reaching a peak around mid to late lactation, and declined thereafter in glands from post-lactating animals. At mid-lactation, the increase in GPT activity, immunoreactive protein, and GPT mRNA relative to the virgin stage was 9.5-, 3.3- and 5.4-fold respectively, on a per cell basis. These data suggest possible transcriptional and post-transcriptional modulation of GPT gene expression during development of mouse mammary gland. The results on hormonal regulation of GPT in mouse mammary explants and primary mouse mammary epithelial cells showed that all three parameters cited above were stimulated maximally by the combined presence of insulin, hydrocortisone, and prolactin, indicating that the hormonal regulation of GPT expression is also mediated at the level of RNA.

Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-dolichol mannosyltransferase.

Glc3Man9GlcNAc2-P-P-Dol serves as the major precursor for the biosynthesis of asparagine-linked glycoproteins in eukaryotes. The first 5 of the 9 mannosyl residues during the assembly of the oligosaccharide moiety within the dolichol cycle in the endoplasmic reticulum are incorporated directly by the action of GDP-Man-requiring mannosyltransferases while the remaining last 4 mannosyl residues are transferred by Man-P-Dol-requiring enzymes. In an earlier study (Shailubhai, K., Illeperuma, C., Tayal, M., and Vijay, I. K. (1990) J. Biol. Chem. 265, 14105-14108), we identified the enzyme UDP-Glc:Dol-P glucosyltransferase by photolabeling rat mammary microsomes with 5-N3-[beta-32P]UDP-Glc. Applying a similar strategy, GDP-hexanolamine-125I-azidosalicylic acid, an analog of GDP-Man, was found to photolabel two polypeptides of 37 and 69 kDa among the microsomal proteins of the rat mammary gland. A differential ammonium sulfate saturation (60-80%) of the detergent-solubilized microsomal proteins enriched the 69-kDa polypeptide. Photolabeling of this polypeptide was specifically inhibited by guanine-containing nucleotides and nucleotide-sugars and was associated with a GDP-Man-requiring mannosyltransferase. The mannosyltransferase was purified nearly 16,000-fold and shown to contain the 69-kDa polypeptide. The purified enzyme catalyzes the transfer of [14C]Man from GDP-[14C]Man to Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol in alpha 1,3-linkage to give [14C]Man alpha 1-->3Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol as the product. Antibodies raised against the 69-kDa polypeptide removed the enzymatic activity from the detergent extract of the rat mammary microsomes and reacted specifically with a polypeptide band of the same size on immunoblots. The purified enzyme showed a pH optima of 7.4-7.8, Km approximately 4 microM for GDP-Man, approximately 2-fold activation by phosphatidylcholine, and a strong inhibition by sulfhydryl-selective reagents, N-ethylmaleimide and p-chloromercuribenzoate. The availability of the highly purified enzyme and a monospecific antibody should allow its molecular cloning for investigating the regulation of the machinery for protein N-glycosylation upon hormonally modulated growth and differentiation of the mammary gland during its ontogeny.

A method for the high efficiency of water-soluble carbodiimide-mediated amidation.

Water-soluble carbodiimides are widely used for carboxyl-amine conjugation. However, extremely variable and low yields, obtained under a variety of conditions, have been a serious problem in the coupling. A simple method, optimizing various parameters of the coupling reaction, in which N-hydroxysuccinimide is included to assist the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-catalyzed amidation reaction is described. A product yield of up to 90% is routinely achieved.

Structure and organization of mouse GlcNAc-1-phosphate transferase gene.

The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, was isolated and characterized. Southern blot analyses demonstrated a single copy gene for GPT. The gene spans about 7.5 kilobase pairs of DNA and is divided into 9 exons by 8 introns. All the introns are found in the coding region, and most of them occur in segments separating the putative membrane-spanning domains. The exon/intron organization of the gene also correlates with the presence of several highly conserved regions of potential functional importance among yeast, leishmania, hamster, and mouse enzymes. Primer extension and reverse transcription-polymerase chain reaction analyses suggested the presence of several potential transcription start sites, with the closest one being approximately 200 base pairs upstream from the translation initiation codon. The 5'-flanking region lacks a typical TATA box, but is high in GC content and contains two putative Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 3'-end reverse transcription-polymerase chain reaction analysis indicated that the first of the two polyadenylation sites was used predominantly, in agreement with a approximately 2.0-kilobase pair GPT message seen on Northern blots of RNA from a wide variety of mouse tissues. This is the first report of cloning of a gene for an enzyme of the dolichol cycle in higher eukaryotes. A novel finding of this study is the observation of a G-->A change between the genomic sequence and nucleotide 280 in the cDNA. This could have important implications as an RNA editing mechanism for regulating the expression of the gene and therefore, protein N-glycosylation. A previous study (11) had shown that the activity of GPT was developmentally regulated in mouse mammary gland, with possible involvement by the hormone prolactin. The availability of the GPT gene with its promoter should facilitate future studies on delineating the mechanism for the hormonal regulation of GPT.

Conserved structural features in glycoprotein processing glucosidase I from several tissues and species.

Glucosidase I initiates the processing of the oligosaccharide, Glc3Man9GlcNAc2, in newly assembled glycoproteins by excising the distal alpha 1,2-linked glucosyl residue in the oligosaccharide. Earlier, the enzyme purified from the ER of rat and bovine mammary gland has been found to have M(r) of 85 kDa, as examined by SDS-PAGE along with a domain structure in which a 39 kDa lumenally-oriented region is anchored to the ER through a transmembrane segment and a short cytoplasmic tail. These studies were further extended to include the enzyme from several different tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. Using anti-rat glucosidase I antibody as a probe and several biochemical parameters such as SDS-PAGE analysis, trypsin-catalyzed digestion, ConA-binding, endo H susceptibility and peptide mapping analysis by cleavage of the tryptophanyl peptide linkages within the enzyme, it was found that glucosidase I in all of the tissue sources examined has an M(r) of 85 kDa and is cross-reactive to anti-rat glucosidase antibody. The enzyme is a high mannose glycoprotein, and has domain features in its structure; the enzyme from mouse, rat, guinea pig and bovine mammary glands and sheep liver is sequentially cleaved by trypsin to generate fragments of 69, 55 and 39 kDa. The rate of release of the different fragments differs for different sources, indicating some evolutionary changes in its primary structure. The trypsin-released fragments from pig kidney enzyme are 69, 45 and 29 kDa in size, identical to the same observed earlier for pig liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing.

Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.

Role of sulfhydryl groups in the function of glucosidase I from mammary gland.

Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain.

We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.

Studies on hormonal modulation of asparagine-linked glycoprotein biosynthesis in explant cultures of rat mammary gland.

Glucosidase I has been purified to homogeneity and polyclonal antibodies against the enzyme have been prepared. The anti-glucosidase I antibodies recognized a single band of 85 kDa on western blot at a dilution as high as 1:2000 and also inhibited the enzyme activity, suggesting the specificity of the antibodies. Con A-Sepharose binding experiment indicates that this enzyme itself is a high mannose type N-linked glycoprotein. The increase in the electrophoretic mobility of 85 kDa band following digestion with endoglycosidase H and F strengthened this observation. The presence of any O-linked sugar attached covalently to glucosidase I could not be detected by binding assays with O-linkage specific biotinylated lectins. The studies on developmental regulation suggest that the synthesis of glucosidase I is modulated with the ontogeny of the gland. Lactogenic hormones, viz. insulin, hydrocortisone and prolactin, appeared to regulate the synthesis of glucosidase I. The possible role of these hormones in the overall regulation of protein N-glycosylation has been discussed.

Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland.

UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.