Jack Pensky

Hereditary Angioneurotic Edema: Two Genetic Variants

Serums of patients with hereditary angioneurotic edema lack inhibitory activity against the esterase derived from the first component of complement. In one group of patients this lack appears to result from failure to synthesize the esterase inhibitor of the first component of complement, whereas in another group of patients an abnormal, nonfunctional protein is synthesized.

Measurement of Circulating Desialylated Glycoproteins and Correlation with Hepatocellular Damage

Addition of increasing amounts of (125)I-labeled desialylated thyroxine-binding globulin (DTBG) to hepatic cell membranes resulted in a progressive increase in binding. Saturability of membrane sites was indicated by a concentration beyond which further increases in [(125)I]DTBG resulted in no further binding. The binding curve for [(125)I]DTBG was similar to binding curves of desialylated orosomucoid, fetuin, and ceruloplasmin. An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG. Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit [(125)I]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 mug DTBG. Patients with Laënnecs cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum. Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of inhibitory activity in the low molecular weight area was also seen. Inhibition of desialylated glycoprotein binding to liver cell membranes by serum from patients with hepatocellular disease raises the possibility that desialylated serum glycoproteins accumulate in the circulation and that patients with compromised hepatocellular function may no longer be able to clear them from the circulation. Alternatively, accumulation of desialylated glycoproteins in the circulation could result from defective protein synthesis by the diseased liver.

Studies on thyroxine-binding globulin (TBG): II. Separation from human serum by affinity chromatagraphy☆

Abstract Thyroxine-binding globulin (TBG) has been separated from human serum by affinity chromatography using Sepharose to which l -thyroxine had been covalently attached. Further purification of the TBG in the material eluted from the T4-Sepharose was carried out by DEAE-Sephadex column chromatography. The final product produced a single stained band on analytic disc-gel electrophoresis. Radio-autography of this material to which tracer amounts of radioactive thyroxine had been added prior to electrophoresis on cellulose acetate showed a single band of thyroxine radioactivity with the electrophoretic mobility of TBG in normal human serum. The calculated yield of TBG by this procedure was 18–37%.

AGGRAVATION BY PROPRANOLOL OF HYPERGLYCAEMIC EFFECT OF HYDROCHLOROTHIAZIDE IN TYPE II DIABETICS WITHOUT ALTERATION OF INSULIN SECRETION

14 hypertensive men with type II diabetes sequentially received, in random order, hydrochlorothiazide 50 mg twice a day, propranolol 80 mg twice a day, and both drugs in combination. The 3-week treatment periods were separated by a 1-week washout period. Hydrochlorothiazide significantly increased fasting glucose by 31% (p less than 0.05) and glycosylated haemoglobin (HbA1c) by 6.0% (p less than 0.10). A similar treatment period of propranolol 80 mg twice a day caused no significant increases. However, when both drugs were taken in combination, fasting glucose rose by 56% and HbA1c by 14.7% (p less than 0.01). The hyperglycaemic effect of hydrochlorothiazide and its potentiation by propranolol were independent of serum potassium and of endogenous insulin secretion as measured by urine C-peptide excretion. The combination of hydrochlorothiazide and propranolol thus seems to cause serious disturbances in glycaemic control in type II diabetics by mechanisms independent of insulin secretion.

Studies on human thyroxine-binding globulin: VIII. Isoelectric focusing evidence for microheterogeneity of thyroxine-binding globulin

Abstract Highly purified thyroxine-binding globulin from pooled human serum homogeneous by conventional criteria, subjected to isoelectric focusing in polyacrylamide gels in a pH gradient from 3–6, produced a pattern of at least nine stainable protein bands. All of these bands appeared to bind thyroxine. Completely desialylated thyroxine-binding globulin subjected to isoelectric focusing produced the same number and pattern of bands located at a different area in the pH gradient. Thyroxine-binding globulin purified from the serum of a single donor was subjected to isoelectric focusing. This thyroxine-binding globulin had the same pattern of protein bands with the exception that one of the major bands seen in the thyroxine-binding globulin from pooled serum was absent. Several possible explanations for these phenomena are discussed.

Studies on thyroxine-binding globulin (TBG): III. Some physical characteristics of TBG and its interaction with thyroxine

Thyroxine-binding globulin (TBG) purified from human serum by affinity chromatography had a mol wt of 63,000–65,000, a sedimentation constant of 3.4S, and E 1 cm 1% at 280 mμ of 6.9. The purified TBG bound thyroxine (T 4 ) in a molar ratio of approximately 1:1. Binding of T 4 to TBG was maximal over a pH range of 6.4–10.4. Below pH 6.4 the T 4 -TBG interaction began to decline and at pH 4.2 binding nearly disappeared. Purified TBG treated with 8 m urea migrated as a single band with a slower electrophoretic mobility than the native protein and did not bind T 4 . After removal of urea by dialysis, five to six protein bands were detectable by polyacrylamide disc gel electrophoresis. The most anodal band had the same mobility as the native protein and regained the ability to bind T 4 , but none of the slower moving bands bound T 4 .

Partial Purification of Plasma Thromboplastin Antecedent (Factor XI) and its Activation by Trypsin

A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca(3)(PO(4))(2) adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185,000) and was specifically inhibited by an antiserum directed against activated PTA. These data suggested that PTA can be converted into its active form by trypsin. PTA was not activated by thrombin, chymotrypsin, papain, ficin, plasmin, plasma kallikrein, tissue thromboplastin, or C. Trypsin converted PTA to its active form enzymatically. Whether trypsin serves to activate PTA in vivo is not yet clear.

Mechanism of acetoin synthesis by α-carboxylase

Abstract 1. 1. Highly purified preparations of α-carboxylase from wheat germ catalyze the synthesis of acetylmethylcarbinol (AMC) from pyruvate and acetaldehyde and from acetaldehyde alone. 2. 2. The AMC thus synthesized has been isolated and its identity established by conversion to diacetyl, periodate degradation, and by analysis of the semicarbazone. 3. 3. It has been shown that the decarboxylase and AMC-synthesizing activities are associated with the same enzyme, α-carboxylase. The conclusion is based on the constant ratio of decarboxylation to AMC synthesis throughout a 2,700-fold purification, on the identical effects of partial inactivation and of pH, and on the identical requirements for diphosphothiamin and a metallic ion for the various activities. 4. 4. The AMC synthesized by α-carboxylase consists of 72% of the (+) and 28% of the (−) optical isomer. 5. 5. A scheme has been proposed for the enzymic transformation catalyzed by α-carboxylase. Known facts about the α-carboxylase of yeast, higher plants, and of animal tissues may be explained in terms of the formation of a relatively stable enzyme-acetaldehyde complex, wherein the aldehyde is linked to the diphosphothiamin moiety of the enzyme. It has been suggested that the formation of a similar intermediate may be the first step in the enzymic oxidation of pyruvate.

Thyroxine-binding globulin: characterization of the binding site with a fluorescent dye as a probe.

The fluorescent dye 1,8-anilinonaphthalenesulfonate competed with thyroxine for binding to thyroxine-binding globulin. Fluorescence analysis indicated that the dye bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23�C) of 4.19 x106M-1, and that thyroxine bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23�C) of 2.35x1010M-1. Displacement of globulin-bound dye by thyroxine was shown by fluorescence quenching, and displacement of globulin-bound thyroxine by dye was demonstrated by ultrafiltration.

Studies on Human Thyroxine-Binding Globulin: VI. THE NATURE OF SLOW THYROXINE-BINDING GLOBULIN

A model system utilizing a highly purified partially desialylated thyroxine-(T(4)) binding protein (STBG) was studied. STBG was prepared by the same affinity chromatographic method we have reported for preparation of highly purified T(4)-binding globulin (TBG). The necessary prerequisite for preparation of STBG was the use of T(4)-substituted Sepharose which had been repeatedly exposed to large volumes of serum for purification of TBG. STBG moved more slowly on cellulose acetate electrophoresis than TBG but had the same molecular weight and antigenic determinants as TBG. It bound T(4) with a 1: 1 molar ratio but its affinity for T(4) was about 10 times less than that of TB. STBG had about onefourth the sialic acid content of TBG and the electrophoretic mobility of this protein was similar to that of a T(4)-binding protein with a mobility slower than that of TBG which has been seen in the electrophoretic patterns of some normal human serums and in serums of patients with hepatic cirrhosis and which does not appear to be an artifact caused by storage and freezing of serum. This fourth slowly migrating T(4)-binding region in electrophoretograms of cirrhotic serums is completely abolished by prior incubation with rabbit antiserum to TBG. The in vitro production of partially desialylated TBG from T(4)-Sepharose which had been previously exposed to large volumes of serum may be due to adsorption of neuraminidases to the Sepharose either directly from serum or as the result of bacterial contamination. Partial desialylation of TBG in vivo may be an early step in the catabolism of this protein.