In vitro preparation of nonenzymatically glucosylated human transferrin, alpha 2-macroglobulin, and fibrinogen with preservation of function.

Human transferrin, alpha 2-macroglobulin, and fibrinogen were incubated with [3H]-glucose. After a 7-day, 37 degrees C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; alpha 2-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 of glucose/mol, or approximately 14 mumol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a Kd = 33 nM and receptor number of 3.4 X 10(5) receptors/cell; nonglucosylated transferrin with a Kd = 31 nM and receptor number of 3.9 X 10(5) receptors/cell. Glucosylated and nonglucosylated alpha 2-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of 125I-labeled, methylamine-treated alpha 2-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated alpha 2-macroglobulin, t1/2 = 3 min. alpha 2-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a Kd of 2.5 nM and receptor activity of 370 fmol/mg cell protein. alpha 2-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a Kd of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced nonenzymatic glucosylation of human serum albumin in diabetes mellitus.

Use of an ion exchange chromatographic method and a colorimetric method with thiobarbituric acid showed that levels of nonenzymatically glucosylated serum albumin were increased in patients with poorly controlled diabetes mellitus compared to controls. The two methods correlated well (r = 0.99) and clearly discriminated between normal and poorly controlled diabetic populations. The levels of glycosylated hemoglobin were also measured in both populations. Several patients apparently in good control based on glycosylated hemoglobin measurements were found to have increased levels of glycosylated albumin. Because albumin has a shorter circulating half-life than does the human erythrocyte, the plasma concentration of glucosylated albumin should be expected to reflect short-term control of hyperglycemia in diabetes. The studies reported here suggest that the level of glucosylated albumin may indeed be a sensitive indicator of moderate hyperglycemia and of early glucose intolerance.

Beta-Adrenergic receptors of human lymphocytes are unaltered by hyperthyroidism.

Lymphocytes from 12 patients with untreated hyperthyroidism were compared to lymphocytes from age- and sex-matched euthyroid control subjects to test the hypothesis that alterations in beta-adrenergic response mechanisms occur in human hyperthyroidism. The binding of (-)[3H]dihydroalprenolol, a compound previously shown in these cells to label binding sites having the characteristics of beta-adrenergic receptors, was assayed and no significant difference was found between the two groups. In addition, the accumulation of cAMP in response to isoproterenol was determined by RIA and, again, no difference was found.

Persistence of MB isoenzyme of creatine phosphokinase in the serum after minor iatrogenic cardiac trauma. Absence of postmortem evidence of myocardial infarction.

The specificity of serum CPK-MB for acute myocardial infarction was examined by retrospective analysis of 401 consecutive patients admitted to Coronary Care Unit over a three and one-half year period with suspected infarction in whom the isoenzyme was subsequently detected. Four patients (1 per cent) who died during the hospital admission had no autopsy evidence of acute myocardial infarction. All four had experienced mild iatrogenic cardiac trauma, following which serum CPK-MG persisted for at least 24 hours. In one patient, a permanent pacemaker had been inserted by the transmediastinal approach. Two patients had been subjected to closed chest cardiac massage and intracardiac puncture, and one to external cardiac massage alone. The findings suggest that persistent identification of serum CPK-MB, although specific for myocardial necrosis, cannot be regarded as diagnostic of myocardial infarction. The implications of this are important to treatment of patients after cardiopulmonary resuscitation and operative trauma to the heart.

Specific photoaffinity labeling of the adenosine 3':5'-cyclic monophosphate receptor in intact ghosts from human erythrocytes.

[(3)H]N(6)-(Ethyl 2-diazomalonyl)-adenosine 3':5'-cyclic monophosphate is incorporated into intact ghosts from human erythrocytes on photolysis at 253.7 nm. Incorporation is blocked in the presence of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and does not occur in the absence of irradiation. Sodium dodecyl sulfate disc gel electrophoresis of solubilized ghosts shows that one protein is labeled. The position of this protein on the gel corresponds exactly to that previously found. [J. Biol. Chem. 247, 8145 (1972)] for the endogeneous protein substrate of the endogenous, cyclic AMP-dependent, protein kinase.

Cyclic adenosine 3':5'-monophosphate-stimulated phosphorylation of isolated neurotubule subunits.

The possible relationship between cyclic adenosine 3':5'-monophosphate (cAMP) and neurotubules in synaptic transmission has been explored. The neurotubular subunit protein from bovine cerebral cortex has been prepared. The addition of cAMP to this preparation in the presence of ATP stimulates the phosphorylation of serine residue(s) in the principal component of the preparation. The neurotubule subunit thus serves as a substrate for an intrinsic, cyclic nucleotide-dependent protein kinase closely associated with the neurotubule subunit. The significance of this finding is discussed in terms of a general model for cellular secretion involving microtubules, cyclic AMP, protein kinase, and calcium ion.