Leonard Jarett

Secretion of various endocrine substances by ACTH-secreting tumors--gastrin, melanotropin, norepinephrine, serotonin, parathormone, vasopressin, glucagon.

Six cases of ACTH‐secreting tumors—which are shown to secrete additional hormonal substances—are reported. The high incidence of this phenomenon among our 15 observed cases with ACTH‐secreting tumor suggests that poly‐humoral secretion in ACTH‐secreting tumors is more common than previous reports would indicate. Although several of our cases are unique in that the particular combination of endocrine substances produced has not been previously reported, the tumor types (islet‐cell carcinoma, carcinoid, pheochromocytoma, oat‐cell carcinoma) are in general those which may be endocrine active in a variety of ways. Ectopic corticotrophin production arises principally in tumors of endocrine tissue, neuroendocrine tissue, endocrine dependent tissue and in oat‐cell carcinoma of the thorax. The possibility that oat‐cell carcinoma of the lung and mediastinum may be a more malignant variety of carcinoid tumor is not proven but should not yet be discounted.

A sensitive and precise isotopic assay of ATPase activity.

Abstract A manual ATPase assay which measures the release of 32Pi from [γ-32P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate 32Pi from [γ-32P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10−15 to 10−7 mol of 32Pi from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-32P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.

Nonuniform Distribution and Grouping of Insulin Receptors on the Surface of Human Placental Syncytial Trophoblast

These studies were designed to investigate the cytologic localization and topographic distribution of insulin receptors in human placental villi. Biochemical studies showed placental villi to specifically bind l25I-insulin. Radioautographic studies showed the specific binding to be localized to the surface of the syncytial trophoblast. Topographic distribution of insulin binding was determined with ferritin-insulin. Initial studies using ferritin-insulin containing some oligomers of ferritin revealed the insulin receptors to be specifically associated with the glycocalyx region of the surface membranes of microvilli. No insulin receptors were detectable in association with the intermicrovillous plasma membrane even though its glycocalyx is in direct continuity with the glycocalyx of microvilli. Monomeric ferritin-insulin showed the same nonuniform distribution of the insulin receptor, which suggests that there is not complete freedom of lateral mobility of the insulin receptors in the surface membrane of this tissue. The insulin receptors were found to occur as singletons or in groups of two or more. Incubations with monomeric ferritin-insulin at 4δ or with tissue prefixed with formaldehyde showed that the groups of insulin receptors were naturally occurring, i.e., they are present prior to and independent of insulin binding and thus not secondary to ligand-induced aggregation. The physiologic meaning of the nonuniform distribution and the groups of insulin receptors is unclear at present.

Nucleotide inhibition of adenyl cyclase activity in fat cell membranes.

Abstract A reproducible assay for adenyl cyclase activity in an isolated fat cell membrane preparation is described. Enzyme activity was stimulated by epinephrine and sodium fluoride, but not affected by insulin. GTP, GDP, GMP, CTP and UTP inhibited the adenyl cyclase activity of the fat cell membrane, but guanosine and cyclic GMP had no effect. In contrast to the inhibitory effect on the fat cell membrane enzyme, GTP did not affect either adrenal or kidney adenyl cyclase activity.

The ability of insulin to alter the stable calcium pools of isolated adipocyte subcellular fractions.

Abstract The calcium contents of adipocyte plasma membranes, mitochondria and microsomes (endoplasmic reticulum) were measured by atomic absorption. Ruthenium red (RR) and EDTA were used to selectively modify post-homogenization artifacts. Values obtained in the presence of RR appear to approximate the in vivo calcium content for mitochondria. Measurements in the presence of EDTA represented a highly stable pool of calcium for all organelles. Insulin (100 micro-units/ml) treatment of adipocytes prior to fractionation caused a conversion of a significant portion of the stable calcium pool to an exchangeable form in mitochondria without altering the total mitochondrial calcium content. Insulin did not affect the calcium content of the plasma membranes. The hormone did significantly increase the calcium content of the microsomes, which probably reflected the increased ability of microsomes isolated from insulin-treated adipocytes to bind and accumulate calcium.

Isolation from Rat Adipocytes of a Chemical Mediator for Insulin Activation of Pyruvate Dehydrogenase

Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity. Phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to block this stimulation. Involvement of the kinase in this activation mechanism was eliminated by the fact that, in the presence of ATP, (1) NaF completely blocked the stimu ation of pyruvate dehydrogenase by the active fraction, and (2) the stimulation of pyruvate dehydrogenase by dichloroacetic acid, akinase inhibitor, was add itive to the stimulation caused by the active fraction. This active fraction may contain an intracellular chemical mediator or second messenger for insulin.

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum.

Abstract The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum ( D. E. Bruns, J. M. McDonald, and L. Jarett, 1976 , J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca2+ + Mg2+-ATPase) of relatively low activity. Mg2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca2+ + Mg2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent Km values of 115 μ m for ATP, 333 μ m for magnesium, and 200 μ m for calcium. Maximum Mg2+-ATPase activity was obtained with no added calcium and 1 m m magnesium. Potassium was found to inhibit Mg2+-ATPase activity at concentrations greater than 100 m m . The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μ m , respectively. (Ca2+ + Mg2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 m m and maximum activity reached at 100 m m potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase are similar to those of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.

A rapid microtechnique for the preparation of biological material for the simultaneous analysis of calcium, magnesium and protein

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r2) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.

Direct effect of insulin on the labeling of isolated plasma membranes by [γ32P] ATP

Abstract Labeling of isolated plasma membranes from rat adipocytes by [γ 32 P] ATP was studied and the effect of insulin on this process was determined. Labeling was bi-phasic, increasing to a maximum while the supply of ATP was sufficient, then decreasing as the supply of ATP was depleted by hydrolysis. Insulin added directly to the assay mixture without pre-incubation decreased the level of labeling of plasma membranes at each time of incubation tested, from 30 sec to 10 min. The effect was insulin dose-dependent and was not observed with the inactive insulin derivative, desoctapeptide-insulin.

Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8--11.5 nmol Ca2+/mg protein after 3--4 min of incubation. The rate of Ca2+ transport was iM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent Km values of 100 muM and 7 muM for ATP concentration ranges above and below 50 muM, respectively. The apparent Km values for Mg2+ and Ca2+ were 132 muM and 0.36--0.67 muM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (Ca2+ + Mg2+)-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity was K+ greater than Na+ = NH4+ greater than Li+. Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity were both inhibited 10--20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 muM p-chloromercuribenzene sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a (Ca2+ + Mg2+)-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution.