Alkaline Ribonuclease Inhibitor in Human Thyroid Roger 1. Greif

and Elizabeth

Thyroids of goitrogen-treated rats contain increased amounts of a protein inhibitor of ribonuclease activity at pH greater than 7 (alkaline ribonuclease inhibitor, ARI). We report here that thyroids from hyperthyroid patients contain more ARI than normal human thyroids. This increase parallels RNA concentration. The inhibitor is heat labile, inactivated by sulfhydryl blocking agents, and has a molecular weight near 50,000 daltons. ARI is quantitated by its

F. Eich

activity against bovine pancreatic RNase, but it also inhibits human thyroid RNase. Analyses of a solitary toxic nodule and its surrounding suppressed tissue confirm in tissues from a single patient our results in tissue from numbers of thyrotoxic and euthyroid individuals and decrease the likelihood that changes are induced by antithyroid medication. A possible regulatory role for ARI is suggested.

R

IBONUCLEASE ACTIVITY at pH 7 or above (alkaline ribonuclease) has been found in many mammalian tissues. ’ This activity is usually masked by the presence of an inhibitor (ARI). The AR1 of rat liver is a protein with an approximate molecular weight of 50,000 daltons which is active against bovine pancreatic RNase (EC 2.7.7.16), is inactivated by heat and sulfhydryl (SH) blocking agentq2 and has been purified by salt fractionation and affinity chromatography.3 AR1 is found largely in cytosol, and since ribosomes contain ribonuclease,4 polysomes cannot be prepared from rat liver unless cytosol is present, presumably to protect RNA from the action of ribonuclease.5 We have reported that the AR1 of rat thyroid is increased by thyrotropin (TSH) stimulation of the gland and decreased when the stimulus is removed.6 In this paper we report our studies of AR1 in the human thyroid. MATERIALS

AND

METHODS

Thyroid specimens were obtained only from normal and hyperthyroid individuals with relatively uniform tissue on microscopic examination. Patients with hyperthyroidism were receiving antithyroid medication and iodine prior to surgery. The normal thyroids were obtained from patients having neck surgery for nonthyroidal disease. Specimens were obtained as soon as possible after surgical removal and stored in liquid nitrogen until immediately before study. Tissue samples were thawed, weighed, and homogenized in 10s; dilution in a buffer containing0.05 M P04, pH 7.4, 0.015 M EDTA, and lo-’ M dithiothreitol using a Polytron homogenizer PT-10. The instrument was run at one-half speed for two 5-set bursts separated by 2 min, with the sample in an ice bath. For inhibitor assay, a diluted homogenate or clarified extract prepared by centrifugation of the homogenate at 105,000 g for 120 min could be used interchangeably. For chromatography, only the extract was used. AR1 was measured by its effect upon crystalline bovine pancreatic RNase as previously described.6 The unit was micrograms of pancreatic RNase 507; inhibited per amount of thyroid

From the Department of Physiology, Cornell University Medical College, New York, N. Y. Receivedfor publication June 23, 1976. Supported in part by Grant GB-31109 from the National Science Foundation. Reprint requests should be addressed to Dr. Roger L. GrieJ Department of Physiology, Cornell University Medical College, 1300 York Avenue. New York, N. Y. 10021. 0 1977 by Grune & Stratlon. Inc. Metabolism ISSN 0026-0495.

Metabolism, Vol. 26, No. 8 (August), 1977

851

GRElF

852

AND EKH

based upon wet weight of tissue, thyroid RNA, or thyroid DNA. The confidence limits of the 50”” inhibition point were estimated as suggested by Goldstein.’ Thyroid RNase was determined in the

presence of the SH blocking agent parachloromercuriphenylsulfonic acid (PMPS) I.5 x lO-4M. DNA was measured by the method of Wollman and Breitman’ using small fragments of thyroid rather than homogenates. A standard curve using calf thymus DNA was included in each series of determinations. RNA was determined by the method of Fleck and Begg,’ and protein by the method of Lowry et al.” using bovine serum albumin as standard. Calf thymus DNA and pancreatic RNase were purchased from Sigma, yeast RNA (highly polymerized) and PMPS from Calbiochem, and Sephadex G-200 and G-100 from Pharmacia. An Amicon PR-8 microfiltration apparatus with UM-IO filter was used to concentrate eluates.

RESULTS

The data from five hyperthyroid patients and seven normals are shown in Table 1. The concentration of RNA per gram of thyroid is doubled in the hyperthyroid patients. Since the amount of inhibitor per gram is also doubled, the inhibitor level per milligram RNA is not different in the two groups. There is a small increase in both the DNA concentration and inhibitor concentration per milligram DNA in the tissue from hyperthyroid patients. The mean values seen in Table 1 were obtained by pooling results from several normal and hyperthyroid individuals. Only the hyperthyroid patients received treatment with antithyroid drugs and supplemental iodine prior to surgery. The determinations on a single patient with hyperthyroidism due to a “hot nodule” are therefore of special interest. This patient, who also received antithyroid medication and supplemental iodine, had a 757: 24-hr “‘I uptake, which by scan was almost entirely localized in the nodule. The surrounding tissue appeared suppressed. As seen at the bottom of Table 1, the results on this patient confirm the findings on the patient pools, with a marked increase in as compared to the surrounding RNA and ARI/g tissue in the “hot nodule” tissue. The markedly lowered RNA level in the suppressed tissue accounts for the increased ARI/mg RNA observed. Inhibitor is present in both normal and suppressed tissue. RNase levels per gram in tissue from hyperthyroid patients did not differ from normal (not shown). Separate experiments showed that the Table 1. Analyses

of Thyroid Tissue Prom Euthyroid

Euthyroid (7) Hyperthyroid

(5)

Patients

Inhibitor Units (10 Arrays per Gland) Per Gram Wet Wt Per mg RNA Per mg DNA

DNA @g/g)

RNA @g/g)

and Hyperthyroid

1.05zk0.11*

1.84 f 0.17*

2.15

zt 0.29t

2.09

zt 0.26*

1.18 * 0.16*

2.10

2.23

4.19

zt 0.32

2.04

f 0.24

1.98 zt 0.30

0.025

f O.l7t(lO)

1.35

1.49

2.87

0.78

i

0.12

f 0.21

Statistical Significance of Differences Between Groups P

0.05 1.99 f 0.05*(s)

2.97

2.00

1.55 i

* 0.02

(4)

0.24

tissue

Alkaline ribonuclease inhibitor in human thyroid.

Alkaline Ribonuclease Inhibitor in Human Thyroid Roger 1. Greif and Elizabeth Thyroids of goitrogen-treated rats contain increased amounts of a prot...
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