Purification and characterization of acute phase rat plasma thiostatin.

Thiostatin was purified from acute phase plasma of turpentine-treated rats by a novel, single-step carboxymethyl-papain Sepharose 4B column chromatographic procedure. Purified thiostatin appeared as a single band in SDS-PAGE with an estimated molecular weight of 68,000. Western blot with polyclonal rabbit anti-thiostatin IgG confirmed a homogeneous immuno-reactive 68 kDa species. Specific activity, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.972 mg kininogen equivalent per mg protein. The yield of thiostatin exceeded 60% and the protein was purified 10.7-fold.

Human oviductal fluid proteins. V. Identification of human oviductin-I as alpha-fetoprotein.

OBJECTIVE: To investigate whether human oviductin-I (hOV-I) from human oviductal fluid (hOF) and human alpha-fetoprotein (hAFP) are identical molecules. DESIGN: Comparison of amino acid and carbohydrate composition of hOV-I with that of hAFP. Isolation of hAFP from oviductal fluid of a patient and evaluation of its immunochemical properties in relation to hOV-I and authentic hAFP standard. SETTING: Procedures were performed in an academic research environment. PATIENTS: Human oviductin-I was isolated from a pooled sample of hOF obtained from four patients in a third world country. AFP was purified from a sample of oviductal fluid obtained from a single patient during her periovulatory period. INTERVENTIONS: Human oviductal fluid was collected coincident to elective tubal ligations. MAIN OUTCOME MEASURES: The amino acid and carbohydrate composition of hOV-I was determined. AFP was purified from hOF using ion-exchange chromatography and gel filtration. The identity between hOV-I and hAFP from hOF was assessed using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot analysis. RESULTS: The amino acid and carbohydrate composition of hOV-I indicated similarities between hOV-I and hAFP. Both hOV-I and hAFP reacted with mouse anti-hAFP monoclonal antibody and purified polyclonal rabbit anti-hOV-I immunoglobulin G in an identical manner as observed by noncompetitive ELISA. Purified AFP from hOF of a single patient had a molecular mass of 66 kd and was found to be identical with hOV-I by Western immunoblot analysis. CONCLUSIONS: Human oviductin-I and hAFP are chemically similar and immunologically identical molecules. We believe that this finding is the first documentation for the existence of AFP in hOF.

Human oviductal fluid proteins. VI. Correlation between alpha-fetoprotein and serum levels of ovarian hormones.

OBJECTIVE: To determine the relationship, if any, between the ovarian hormonal status of women and levels of alpha-fetoprotein (AFP) in their oviductal fluid. The threshold questions were the following: (1) does the presence of AFP in human oviductal fluid (hOF) represent a generalized phenomenon and (2) if so, is there any relationship between AFP in hOF and the ovarian hormones, estradiol (E2) and progesterone (P)? DESIGN: Eleven women who elected minilaparotomy tubal ligations volunteered to donate hOF and serum samples for this study. AFP, E2, and P were determined be radioimmunoassay. SETTING: Procedures were performed in an academic research environment. PATIENTS: This study encompasses only those patients who elected minilaparotomy tubal ligation and volunteered to donate hOF and sera. Clinical features of these patients are described. INTERVENTIONS: Patients undergoing tubal ligations had Foley catheters inserted into the fimbriated ends of each oviduct, and hOF was collected for 24 hours. Concomitantly, blood was drawn for analyses of serum. The Investigational Review Board of the Erie County Medical Center approved this study requiring the collection of oviductal fluid from human subjects. RESULTS: Levels of AFP in hOF did not correlate with the serum concentrations of E2 and P per se. However, there was a highly significant correlation between levels of AFP in hOF and serum E2:P ratio. The concentration of AFP in hOF progressively increased with respect to the ratio of serum E2:P. Although AFP was present in all samples of hOF, it was undetectable in corresponding sera. CONCLUSIONS: Based on these data, it is concluded that AFP is generally present in hOF. Furthermore, it is suggested that AFP in hOF may be under the control of ovarian steroids.

A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.

Human oviductal fluid (hOF) proteins. IV. Evidence for hOF proteins binding to human sperm.

In the management of infertile patients, it is well recognized that gamete intrafallopian transfer (GIFT) has a higher success rate than in vitro fertilization and embryo transfer. Oviducts produce unique proteins that may be responsible for the success of GIFT. Unique proteins from human oviductal fluid (hOF) have been purified in the authors' laboratory. One of these proteins of 54 kDa molecular weight, and containing carbohydrate, was obtained in a highly purified state. In this study, human sperm were incubated with: (1) a mixture of hOF-specific proteins, and (2) the highly purified hOF glycoprotein, designated human oviductin I (hOV-I). Using indirect immunofluorescence, the authors studied the ability of hOF proteins to bind to human sperm. The mixture of hOF proteins appeared to bind to the surface of the entire sperm. Conversely, hOV-I binding was restricted to the head region only. Studies are in progress to discern the function of the sperm-binding interaction(s) with hOF proteins.

Human oviductal fluid proteins. III. Identification and partial purification.

The role of the macromolecular constituents of human oviductal fluid (hOF) in reproductive physiology is poorly understood. Oviductal fluid contains proteins derived by transudation from serum and those synthesized and secreted by the tubal mucosa. In this communication, we describe purification of hOF-specific proteins. As a first step, serum proteins were removed with an immunoaffinity gel. Fractionation of the proteins unique to hOF was accomplished with DEAE-cellulose chromatography. One of these proteins was obtained in a highly purified state after gel filtration on G-75 Sephadex. This protein of 54 kDa molecular weight had a pI of 4.5 and contained carbohydrate. We propose that this hOF glycoprotein be designated "human oviductin I" (hOV I).

Sugar residues on proteins.

Glycoproteins have become increasingly important in the structure and function of many different mammalian systems; for example, membrane glycoproteins and glycoprotein hormones. It is, therefore, important to understand their chemistry, which would include an understanding of both the carbohydrate and protein parts of the molecule. Since the chemical characterization of the protein moiety has been extensively examined and the techniques for its characterization are well worked out, only the carbohydrate portion of glycoproteins will be reviewed in this article. The chemical nature of the carbohydrate moiety of glycoproteins will be examined. First, the types of monosaccharides present in animal systems, especially those in the mammalian systems, will be described. Next, various types of simple and complex carbohydrate chains will be discussed to establish the diversity, size, and number of chains present in the carbohydrate units in different glycoproteins. Then, the type of linkages of the carbohydrate to the protein will be examined to determine if the primary sequence of protein is important in determining the size and type of carbohydrate chains present in glycoproteins. Finally, the current methods of structural elucidation such as monosaccharide sequence, intersugar bonds, and anomeric linkages in the carbohydrate moiety of glycoproteins will be reviewed. These methods include the techniques of periodate oxidation, methylation, partial acid hydrolysis, and specific glycosidase digestion of glycoproteins, as well as the latest techniques using micromethods of carbohydrate quantitation and characterization involving gas chromatography and mass spectrometry. The function of the carbohydrate in glycoproteins will also be considered. First, hormone glycoproteins will be discussed in their relationship to the immunological and biological function of the glycoprotein when the carbohydrate is sequentially removed. Next, the function of the carbohydrate in the turnover of glycoproteins will be discussed. These topics will be considered in order to develop an understanding of a specific function(s) of the carbohydrate in glycoproteins.

The in vitro inhibition of bovine milk lipoprotein lipase by a glycoprotein preparation from human atherosclerotic intima.

Intimal tissue from human atherosclerotic aortae was collected by the Dermatome procedure. The tissue was extraved with 5 mM Tris.HCl buffer containing 0.3 M NaCl and 1 mM EDTA, pH 7.4. The ammonium sulfate precipitate between 0.4--0.8 saturation obtained from the extract was fractionated on a DEAE-cellulose column and the effluent was monitored for lipoprotein lipase inhibition employing purified bovine milk enzyme. The substrate used was an emulsion of purified olive oil and tritiated triolein. Human serum was the source of activator of the substrate. A peak of inhibitory activity was eluted between 0.15--0.17 M NaCl. The major component in the purified material had properties similar to a glycoprotein (lipolipin) which has previously been purified from porcine aorta and shown to inhibit lipoprotein lipase activity. The partially purified human inhibitor decreased both the basal and the serum-stimulated activity of milk lipoprotein lipase. The inhibition was non-competitive with respect to serum. However, high levels of triglyceride substrate appeared to relieve the inhibitory effect. It is postulated that the inhibitor may be involved in an interaction with the emulsified lipid denying the enzyme access to its substrate.

Purification of jack bean meal beta-D-galactosidase by a new affinity adsorbent.

A simple procedure has been developed for the purification of jack bean beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) by affinity chromatography employing a new affinity adsorbent. The ligand 6-N-beta-(4-aminophenyl)-ethylamino-3-O-beta-D-galactopyranosyl-6-deoxy-L-gulitol was prepared by the reaction between lactose and beta-(4-aminophenyl)-ethylamine and was coupled to cyanogen bromide activated Sepharose 4B via the amino groups of the 4-aminophenyl moiety. This affinity gel resulted in a 111-fold purification of beta-D-galactosidase with a 64% recovery of the enzyme. With p-nitrophenyl-beta-D-galactopyranoside as the substrate the apparent Km and V values were 0.59 mM and 1.87 mumol/min per mg, respectively. The method for purification of beta-D-galactosidase may be applicable to other glycosidases depending upon the choice of specific di- or oligosaccharides of known structures to be used in the preparation of ligands.

Further studies on a highly purified glycoprotein from the intimal region of procine aorta.

Highly purified glycoprotein from the intimal region of porcine aorta was isolated with minor modifications of the procedure described previously. The molecular weight of the glycoprotein as determined by sedimentation equilibrium method either in presence of 0.1 M NaCl or 6 M guanidine-HCl containing beta-mercaptoethanol was 72 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native glycoprotein and its S-carboxyamidomethyl derivative at different acrylamide concentrations showed no difference in the molecular weight indicating the absence of subunits. Attempts to determine the identity of the amino-terminal acid by a dansylation technique indicated that the amino group is not free. The carboxy-terminal amino acid was found to be serine after treatment of the glycoprotein with carboxypeptidase A. The glycoprotein did not contain an alkali-labile (O-glycosidic) carbohydrate-protein linkage as tested by the beta-elimination reaction. The release of monosaccharides from the glycoprotein as a function of time was studied employing mild acid hydrolysis (0.5 M HCl, 80 degrees C) and also by the use of neuraminidase, alpha-D-and beta-D-glucosidases and beta-D-N-acetylglucosaminidase. From the observations of the release of monosaccharides and analogy with standard features determined by other investigators on soluble aortic glycoproteins, a prediction has been made as to the general features of the carbohydrate moiety of the glycoprotein.