Oxygen regulated 80 kDa protein and glucose regulated 78kDa protein are identical.

Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses.

Isolation and characterization of rat alpha-1-antitrypsin.

From 330 ml of rat serum, 222 mg of alpha-1-antitrypsin have been isolated and purified with an overall yield of approximately 20%. The preparation was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sedimentation equilibrium centrifugation, and immunoelectrophoresis. Rat alpha-1-antitrypsin exhibited Mr = 47,000 +/- 1,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 1,000 by equilibrium ultracentrifugation; the sedimentation coefficient (s20,w) was 3.29. Rat alpha-1-antitrypsin exhibited a trypsin-combining ratio (moles of trypsin inhibited/mol of alpha-1-antitrypsin) of 0.88. Rat alpha-1-antitrypsin showed significant differences in amino acid composition when compared to human alpha-1-antitrypsin, particularly in lysine, glutamic acid, arginine, methionine, and tyrosine content. Rat alpha-1-antitrypsin contains 14.3 residues/mol of N-acetylglucosamine, 5.0 residues/mol of mannose, 4.2 residues/mol of galactose, and 5.8 residues/mol of sialic acid. Monospecific antibody produced in a rabbit against our purest preparation of rat alpha-1-antitrypsin does not cross-react immunologically against human, calf, fetal calf, mouse, or chicken sera. The availability of a pure preparation of rat alpha-1-antitrypsin as well as the specific alpha-1-antitrypsin antibody will facilitate studies on the biosynthesis and secretion of this important protease inhibitor in an appropriate animal model.

Effect of growth conditions on the content of the major groups of carbohydrates in chick embryo fibroblasts.

The levels of glycogen, hyaluronic acid, chondroitin sulfates, N-acetylneuraminic acid, all of the monosaccharide components of the glycoprotein and glycolipid fractions, and the monosaccharide pools were measured in cultured chick embryo fibroblasts. Under all growth conditions, the glycogen plus the glucose phosphate pool contained approximately 50% of total monosaccharide content of the cells. However, marked qualitative and quantitative alterations were found in the glycoprotein, glycolipid, and mucopolysaccharide fractions when growing cells reached confluence, when the growth temperature was shifted from 36 to 41 degrees, or when the cells were transformed with Rous sarcoma virus. From 65 to 95% of the total monosaccharide residues in these complex carbohydrates were found in the glycoprotein fraction, while the glycolipids contained only 5 to 10% of the residues, and the mucopolysaccharides contained 5 to 25%. Changes in the complex carbohydrates in normal cells following changes in cell density or growth temperature were so great that they obscured any transformation-dependent changes that might have occurred consistently in the virus-infected cells under different growth conditions.

Turnover of cellular carbohydrates in normal and Rous sarcoma virus-transformed cells.

We have analyzed the distribution of glucosamine-labeled polymers on the cell surface, in the growth medium, and inside the cell and the net turnover of these polymers during the process of malignant transformation of chick embryo fibroblasts by Rous sarcoma virus. The distribution of label and the turnover kinetics for hyaluronic acid, total glycoproteins, and chondroitins were found to be identical in both normal and transforming cultures. We conclude that nonspecific degradative processes are probably not involved in causing transformation-related alterations in cell surface carbohydrates, althougn degradation of specific macromolecules is not excluded.