SP-Sephadex Chromatography Research Articles

Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of l-(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κm values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κi value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κi value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.

Purification and characterization of progesterone-binding globulin in the pregnant Cape porcupine, Hystrix africaeaustralis

Progesterone-binding globulin (PBG) was purified from pooled pregnant Cape porcupine ( Hystrix africaeaustralis) plasma by SP-Sephadex Chromatography, followed by Sephacryl S-200 gel filtration. The final product was homogeneous according to high performance capillary electrophoresis, SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. A molecular mass of 65,300 Da was calculated from sedimentation equilibrium centrifugation data. Scatchard analysis indicated a dissociation constant of 0.82 × 10 −9M, and PBG concentration of 18.4 nmole/mg protein.

Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum

A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery yield was 38%. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5–6.0 and 45–50°C, respectively, when olive oil was used as the substrate. The lipase was stable over a pH range of 4–10 at 30°C for 4 h, and up to 40°C at pH 5.6 for 30 min. Furthermore, the enzyme was not inactivated even after incubation at 30°C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, benzene and ether for 20 h. The activity did not decrease in a reaction with stirring in a mixture containing 50% DMSO or dimethylformamide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6≤C≤12), and cleaved only 1,3-ester bonds of triolein. The enzyme had an N-terminal sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases from F. heterosporum and another strain F. oxysporum.

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1—-3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.

Multiple molecular forms of finger millet subtilisin and amylase inhibitors

Nine varieties of finger millet (Eleusine coracana Gaertn) including a wild form were screened for proteinase and α-amylase inhibitory activities. Subtilisin inhibitory activity was present in all the varieties examined and was highest in the wild form, while the cultivated varieties had more trypsin inhibitory activity. Isoelectric focusing of a 60% ammonium sulphate fraction of the wild form, before and after complexing with subtilisin, showed multiple forms of subtilisin inhibitors one of which was also active against trypsin. Isoforms of finger millet subtilisin inhibitors were isolated from the ammonium sulphate fraction by ion exchange chromatography on DEAE-Sephadex followed by SP-Sephadex chromatography. Copurification of trypsin and α-amylase inhibitory activities with subtilisin inhibitory activity was observed during cation exchange chromatography. PAGE analysis revealed them to be charge isomers with pI in the range 5·5–6·0. SDS–PAGE gave an estimate of 12 000 mol wt for each of them. Finger millet subtilisin inhibitors were more active on bacterial proteinases than on bovine trypsin and chymotrypsin.

Variations of cell wall peroxidases in epicotyls of Cicer arietinum during growth

Fractionation and partial purification of the proteins extracted from cell walls of Cicer arietinum epicotyl reveal the presence of two fractions with peroxidase activity, denominated Px-1 and Px-2 according to their order of elution from a SP-Sephadex chromatography column. The specific activity of the Px-2 fraction increases with the age of the epicotyls, with an inverse relationship to their growth capacity. No such a relationship could be found with the peroxidase activity of the Px-1 fractions. These results indicate that the cell wall peroxidase fraction Px-2 could be related to cell wall stiffening processes. According to this, acid pHs-values, which induce cell wall loosening processes, diminish the enzymatic activity of the Px-2 fraction to a considerable extent.

Changes during epicotyl growth of an autolysis-related β-galactosidase from the cell wall of Cicer arietinum

During the growth of Cicer arietinum epicotyls an increase in specific cell wall β-glucosidase and β-galactosidase activities were observed. In contrast, the amount of cell wall proteins extracted with 3 M LiCl decreased between the third and the fourth day of germination, remaining constant up to the seventh day. Partial purification of the cell wall protein extracts from different days by SP-Sephadex chromatography showed that one fraction increased both in the amount of protein and β-galactosidase activity throughout the growth of epicotyls. This protein fraction, which is the third β-galactosidase (βIII) eluted from a SP-Sephadex chromatography, has been previously characterized by us as the main enzyme involved in the autolytic process.

4] Separation and determination of thiamin and its phosphate esters by SP-sephadex chromatography

This chapter describes the separation and determination of thiamin and its phosphate esters by SP-Sephadex chromatography, which is considered as a reliable and capable method for separating all forms of the vitamin. The thiamin compounds are assayed fluorometrically using the alkaline cyanogen bromide reaction of Fujiwara and Matsui. To one aliquot of the fraction, 0.9 ml of cyanogen bromide is added, followed by 0.6 ml 30% NaOH. To the second aliquot, the order of addition is reversed to destroy thiamin and yield a blank value. The difference between the two readings is used to calculate the thiamin content. With this procedure, 10 pmol of the thiamin compounds could be reproducibly determined: linearity is obtained to 200 pmol. When 200 pmol of authentic radioactive thiamin and its phosphate esters are added to a brain extract, the recovery of the compounds is as: ThTP, 100.5%; ThPP, 98.3%; ThMP, 99.6%; thiamin 98.4%. As the procedure is developed to determine the forms of thiamin that occur in synaptic plasma membranes, the applicability of the procedure is appraised after preparing subcellular fractions of rat brain.

Structural characterization and antitumor activity of the extracts from matted mycelium of cultured Grifola frondosa.

The fruit body of Grifola frondosa, an edible mushroom, is used as a food in Japan. However, the matted mycelium of the fruit body is not utilized, In this paper, structural characterization and antitumor activity of the extracts from the matted mycelium of this fungus were examined. Hot water, cold alkali, and hot alkali extracts of the mycelium all contained polysaccharides ; that in the cold alkali extract was composed of glucose, but those in the hot water and hot alkali extracts contained glucose, mannose and/or xylose. Each fraction showed potent antitumor activity against murine solid tumor, Sarcoma-180. The antitumor activities of the alkali extracts were more potent than that of the hot water extract. By means of DEAE-Sephadex chromatography, α-amylase treatment, and SP-Sephadex chromatography, a neutral β-1, 3-glucan was purified from the cold alkali extract. From the results of nuclear magnetic resonance spectroscopy, methylation, periodate oxidation, enzymic degradation, and physicochemical characterizations, the purified antitumor glucan was concluded to be a β-1, 3-glucan branched at C-6 of every third main-chain glucosyl unit. The structure is quite similar to that of the antitumor glucan obtained from the fruit body of this fungus. It is concluded that the matted mycelium of G. frondosa may be useful as a source of antitumor β-1, 3-glucan.

Proteolytic conversion of insulin-like growth factors to an acidic form(s)

The relative amounts of the various forms of bioassayable insulin-like growth factors (IGF) isolated from human serum or serum fraction Cohn IV-1 depend on the purification procedure. With acid gel filtration or acid/ethanol extraction as the initial step, IGF-II (pI approximately 6.5) was the most abundant (40-70%) followed by somatomedin A (pI approximately 7.4; 15-23%), an acidic form of insulin-like activity (ILA pI 4.8) (13-21%) and IGF-I (pI approximately 8.5; 5-27%). If, however, pH 5.5 ion-exchange chromatography on SP-Sephadex was used prior to acid gel filtration, the acidic pI 4.8 form was the major (greater than 90%) species recovered and was accompanied by a quantitative loss of the other IGF species. This suggested a possible conversion of IGF-I, somatomedin A and/or IGF-II to the acidic ILA pI 4.8 form(s) during the SP-Sephadex procedure. Further experiments indicated that differences in the yields of ILA pI 4.8 were not due simply to differences in the initial pH conditions of the various methods (i.e. acid versus neutral), although exposure to pH 9.7 (a pH experienced during elution of IGF activity from the SP-Sephadex) did appear to play a role. The involvement of the carrier protein in the conversion process was tested by subjecting carrier-free IGF-I and IGF-II to the SP-Sephadex procedure. No conversion of the free forms to ILA pI 4.8 occurred. To examine the possible role of proteinase in the conversion of IGFs to ILA pI 4.8, SP-Sephadex chromatography was performed in the presence of a broad spectrum proteinase inhibitor. The IGF distribution pattern obtained closely resembled the 'normal' pattern seen with acid gel filtration, indicating that proteinase inactivation had prevented conversion to ILA pI 4.8. These data suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to more acidic ILA pI 4.8 form(s) (i) occurs during SP-Sephadex chromatography, (ii) is not prevented simply by prior acid exposure, and (iii) takes place only when IGF-I and -II are in their high-Mr carrier-bound forms. Since IGF-I and IGF-II, although homologous, have unique amino acid sequences, the conversion of both IGFs implies that at least two acidic ILA forms exist. Nevertheless, because ILA pI 4.8 retains the full spectrum of IGF bioactivities in vitro, and significant quantities are present in normal human serum (21%), it would suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to ILA pI 4.8 in vivo may be a physiologically significant event.

Identification of an autostimulatory growth factor in the extracts and conditioned medium of GH3/C14 rat pituitary cells in culture.

An acid- and heat-stable low molecular weight (Mr 2,500-6,200) autostimulatory growth factor has been identified in extracts of GH3/C14 rat pituitary tumors, in extracts of these cells in culture and in their serum-free conditioned medium. The concentrations of autostimulatory activity required to achieve ED50 in serum-free culture were 4.3, 4.0 and 85 micrograms/ml for extracts of tumors, the cultured cells, and from the conditioned medium of the cells in culture, respectively. To characterize this activity, we have used an isolation method that included 0.1 M acetic acid extraction, heating at 95 C, SP-Sephadex chromatography, and finally, Sephadex G-50 chromatography. The identification of this activity if rapidly growing, relatively autonomous GH3/C14 tumors suggests an autostimulatory role in the conversion from estrogen-responsive growth to autonomy.

Heterogeneity of horse transferrin: the role of carbohydrate moiety.

Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electrophoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatography and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N-terminus (glutamic acid followed by glutamine) and the C-terminus (valine). Differences were found in carbohydrate composition between components 2a and 4b. Component 2a contained 10 moles of sugar components per mole of protein (4 hexoses, 4 hexosamines and 2 sialic acids), while component 4b contained twice the number of both total carbohydrates and individual sugar components. Carbohydrates were identified as mannose and galactose (ratio mannose: galactose approximately equal to 1.5:1), N-acetylglucosamine and N-acetylneuraminic acid. At present it is not clear whether the difference between the two components resides solely in the difference of carbohydrate contents. It is proposed that component 2a has one diantennary glycan, while component 4b has two.

Increased levels of new spasmogenic substances released by trypsin from plasma of hypertensive rats.

The present study was undertaken to evaluate the participation of the kallikrein-kinin system in the normalization of blood pressure after release of the clip in one-kidney, one clip hypertensive rats ( 1K1C ). Kininogen was determined before and after unclipping by tryptic digestion of denatured rat plasma, and spasmogenic activity was measured with isolated guinea pig ileum. In contrast to human plasma for which bradykinin (BK) is the only trypsin-releasable spasmogenic substance ( TRSS ), rat plasma contains non-BK TRSS (Fractions P1 and P2) as well as BK. Fractions P1 and P2 were separated from BK by SP-Sephadex chromatography. An increase of total TRSS was demonstrated 60 days after clipping and reached a maximum at approximately Day 75, which was two times that of the normotensive controls (NC). The level of total TRSS did not change after unclipping . The increased level of TRSS in the hypertensive state confirmed the observations of other investigators who reported increased kininogen levels but who could not distinguish between BK and non-BK TRSS because bioassays were performed without prior chromatographic separation of the spasmogenic activities. Fractions P1 and P2 were present in the TRSS of both 1K1C and NC plasma, but were two to six times higher in 1K1C and thus probably accounted quantitatively for the increased TRSS in 1K1C . The data suggest that in the hypertensive state there is an alteration in the relative amounts of some plasma proteins that contain non-BK TRSS within their amino acid sequences. Fractions P1 and P2 also contain potentiating peptides and have not yet been purified to homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Rodent kinin-forming enzyme systems—I: Purification and characterization of plasma kininogen

Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 μg bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelec-trophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).

Haemolytic factor from the crop of Rhodnius prolixus: Evidence and partial characterization

A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.

Separation and characterization of two different species of rat angiotensinogen

Two distinct species of rat angiotensinogen (A-1 and A-2) were purified from plasma of nephrectomized rats by combining ammonium sulfate fractionation, chromatography on Blue Sepharose and SP-Sephadex, gel filtration and preparative polyacrylamide gel electrophoresis. Separation of the two species was accomplished in the SP-Sephadex chromatography step, A-1 eluting before A-2. The two angiotensinogen species had identical electrophoretic mobilities on analytical polyacrylamide gel electrophoresis, but differed in their apparent molecular weights as obtained by SDS-gel electrophoresis (A-1, M r 60000; A-2, M r 56400). In analytical isoelectric focusing each species displayed a characteristic double band with isoelectric points of 4.54 and 4.60 for A-1, and 4.69 and 4.76 for A-2. These physicochemical differences can be accounted for by the difference in carbohydrate content: A-1, when compared to A-2, had a higher content of sialic acid (5.0 and 2.1 mol/mol), neutral hexoses (10.2 and 5.9 mol/mol) and aminobexoses (10.5 and 7.0 mol/mol, respectively). Antiserum raised against rat angiotensinogen crossreacted completely with both angiotensinogens. Both species could also be isolated from plasma of non-nephrectomized rats, which indicates that they may be present under physiological conditions. The physiological significance of the occurrence of these species of angiotensinogen is still unknown.

Purification of a soluble and a wall-bound form of beta-glucosidase from Mucor racemosus.

beta-Glucosidase activity in crude extracts of Mucor racemosus exists in a soluble form and in a wall-bound form which sediments at 3,500 x g. The soluble form and a wall-bound form were purified to homogeneity by ammonium sulfate fractionation. DEAE-Sephadex chromatography, and SP-Sephadex chromatography. Both forms were identical in all parameters measured. Each enzyme is a glycoprotein of 91,000 daltons, with an identical amino acid composition and N-terminal amino acid of lysine; both contain about 10% carbohydrate. Both forms catalyze the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside with identical kinetic constants.

20] Cellobiose oxidase from Sporotrichum pulverulentum

Publisher Summary This chapter focuses on an assay method for the synthesis of cellobiose oxidase from Sporotrichum pulverulentum . The oxidation of the assay substrate, lactose (4- O - β - galactosylglucose), is measured indirectly. The product of the reaction is lactobionic acid, which is quantitatively converted to an equimolar amount of glyoxylic acid. In the presence of lactate dehydrogcnase, glyoxylic acid is reduced to glycolic acid, resulting in the oxidation of nicotinamide adenine dinucleotide dehydrogenase (NADH) and a decrease in the absorbance at 340 nm. The activity of cellobiose oxidasc is determined by the decrease in absorbance at 340 nm. Cellobiose oxidase activity is measured with a modification of the lactate dehydrogenase assay for aldonic acids. The steps involved in the purification procedure are (1) SP-Sephadex chromatography, (2) agarose 0.5 M chromatography, (3) phenyl-sepharose chromatography, and (4) isoelectric focusing. The oxidation of cellobiose by cellobiose oxidase generates superoxide anion. Cellobiose oxidase may oxidize the reducing glucosyl unit exposed by the action of endo-1,4- β -glucanases on cellulose.

Isolation and characterization of the basic proline-rich proteins from rat parotid saliva

Five fractions of basic proline-rich proteins were isolated from rat parotid saliva, obtained by surgical cannulation of the ducts. The purification procedures employed DEAE-Sephadex to isolate a heterogeneous break-through fraction containing the basic proline-rich proteins, followed by gel filtration on Sephadex G-200 to separate the high molecular weight glycoprotein, fraction A, from the other basic proline-rich proteins which were resolved into four additional fractions, SP-1 to SP-4, by ion exchange chromatography on SP-Sephadex. The proteins differed in their amino acid composition and content of neutral and amino sugars. All the proteins were characterized by a high proportion of proline (approx. 40 mol per cent) and glycine (11–23 mol per cent). Four of the fractions were also enriched in glutamic acid glutamine (19–26 mol per cent). The exception was fraction SP-4, which contained lower levels of glutamic acid glutamine and has no counterpart in human basic proline-rich proteins. Fraction A, the basic glycoprotein, was heavily glycosylated (59 mol per cent), whereas SP-2 and SP-4 were less glycosylated. Fractions SP-1 and SP-3 contained low levels of neutral and amino sugars. Basic proline-rich proteins constitute a smaller percentage of the total protein in rat parotid saliva than they do in human parotid saliva (10.5 versus 40 per cent). Rat basic glycoprotein fraction constitutes less than 1 per cent whereas the human glycoprotein fraction constitutes 17 per cent. Rat basic proline-rich proteins appear to be larger and less basic than most of the human basic proteins, and they resolve into fewer protein fractions (4 versus 9) with SP-Sephadex chromatography.