Role of microtubules in the distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies.

Immunofluorescence microscopy reveals that both microtubule organizing center (MTOC) and Golgi apparatus are contained in the same perinuclear area of A549 cells in interphase. The cells display long microtubules stretching radially from the MTOC to the plasma membrane. Treatment of cells with taxol results in polymerization of microtubules without relation to the MTOC and formation of microtubule bundles predominantly localized in the cell periphery. After incubation with taxol, the Golgi apparatus is fragmented and is conspicuously present in areas of the cytoplasm enriched in microtubules. Incubation of cells with Colcemid results in complete depolymerization of microtubules and fragmentation of the Golgi into elements randomly distributed throughout the cytoplasm. Cells treated with taxol before being incubated with Colcemid contain large numbers of Golgi-derived elements in close association with Colcemid-resistant microtubules. Microtubule depolymerization by vinblastine also is followed by fragmentation of the Golgi apparatus. These Golgi-derived elements show no association with the atypical polymers of tubulin induced by vinblastine. The codistribution of Golgi-derived elements with taxol-induced microtubule bundles can be reversed by microinjection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin.     

Role of nucleotides in tubulin polymerization: effect of guanylyl 5''-methylenediphosphonate.

Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.     

Differential turnover of tyrosinated and detyrosinated microtubules.

Turnover of tyrosinated and detyrosinated microtubules ([Tyr]MTs and [Glu]MTs, respectively) was analyzed by the combined use of hapten-mediated immunocytochemistry and peptide-specific antibodies. Cells were microinjected with hapten-labeled tubulin and then processed for triple-label immunofluorescence to determine the pattern of incorporation of the injected subunits into [Tyr]- and [Glu]-MTs. Within 2 min of microinjection, hapten-labeled domains were present at the ends of virtually all [Tyr]MTs but were absent from most [Glu]MTs, demonstrating that [Tyr]MTs grew, whereas most [Glu]MTs did not. After 1 hr of incubation, all [Tyr]MTs analyzed were copolymers of endogenous and hapten-labeled subunits, indicating complete and rapid turnover of these MTs. However, the majority of [Glu]MTs were not hapten-labeled, indicating that they had not turned over. Even 16 hr after injection, cells that had not divided retained a small proportion of [Glu]MTs lacking hapten, implying that some had persisted for most of a cell generation. At mitosis, all MTs were hapten-labeled, indicating that the stable interphase [Glu]MTs had depolymerized. The results establish that the MT network is heterogeneous in its turnover rate, being composed of at least two populations: [Tyr]MTs that turn over rapidly and [Glu]MTs that turn over slowly.     

Role of nucleotides in tubulin polymerization: effect of guanosine 5''-methylene diphosphonate.

Incubation of purified rat brain tubulin with guanosine 5'-methylene diphosphonate [GMP(CH2)P] (1 mM), a GDP analog resistant to hydrolysis, results in the polymerization of 20-30% of the total tubulin present. Analogous incubations with GDP (1 mM) do not result in tubulin polymerization. Polymerization with GMP(CH2)P occurs in the presence of alkaline phosphatase (EC 3.1.3.1) under conditions that completely hydrolyze the likely phosphate donors (GTP, GDP, and GMP) as well as the potential product [GMP(CH2)PP] of the transphosphorylase activity present in purified tubulin preparations. Tubulin polymerization in vitro thus can occur in the absence of gamma-phosphate and phosphate bond hydrolysis at the exchangeable nucleotide-binding site of tubulin. Polymerization of tubulin by GMP(CH2)P is neither prevented nor reversed by concentrations of calcium (2 mM) that prevent microtubule assembly and disrupt already formed microtubules induced by GTP. However, tubulin polymerized with GMP(CH2)P is readily depolymerized by cold (4 degrees, 30 min). The possible involvement of GTP alpha-beta bond hydrolysis must be considered seriously as playing a role in the process of microtubule depolymerization.     

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

We have examined mutant lines of Chinese hamster ovary cells that have increased resistance to the antimicrotubule drug Colcemid. Analysis of the functional properties of purified microtubule protein indicates that increased tolerance to the drug in vivo is reflected in altered properties of microtubules and tubulin in vitro. In this study, we have examined one series of related mutants and have found different microtubule alterations associated with each selection step. These changes include decreased Colcemid-binding affinity, an altered electrophoretic pattern of tubulin subcomponents, increased resistance to Colcemid inhibition of polymerization in vitro and, in one case, a decreased critical concentration for microtubule assembly. Characterized mutants of the class described here will be useful for probing the regulation of microtubule assembly in vivo.     

Dynamic reorientation of cortical microtubules, from transverse to longitudinal, in living plant cells

The direction in which plant tissue cells expand is reflected in the alignment of microtubules in the cortical array. When microtubules and coaligned wall microfibrils are arranged transversely around the cell, turgor pressure is chaneled into cell elongation. However, various agents (such as wounding, ethylene, abscisic acid) can cause the microtubules to reorientate by 90 degrees so that they become aligned parallel to the cell's long axis, allowing lateral expansion instead of elongation. The mechanism by which microtubules undergo rapid shifts of alignment is crucial to understanding growth control in plants, but because current models are derived from studies on fixed cells, nothing is known about the dynamics of converting one microtubule alignment to another. Cells tend to have one predominant microtubule alignment--transverse, oblique, or longitudinal--but it is not established whether each represents a stable independent set that only changes by rounds of complete de- and repolymerization, or whether reorientation is a more continuous process involving movement of stable or dynamic microtubules. By microinjecting pea (Pisum sativum) epidermal cells with rhodamine-conjugated brain tubulin and optically sectioning them by confocal laser scanning microscopy, we could follow labeled microtubules for up to 2 hr as they reorientate. Reorientation does not occur by complete depolymerization of microtubules in one orientation followed by polymerization of a new array in another orientation. Instead, increased numbers of discordant microtubules in nontransverse alignment appear in particular locations. Neighboring microtubules then adopt the new alignment, so that there is a stage during which different alignments coexist before the array on the outer tangential cell face finally adopts a uniform steeply oblique/longitudinal configuration. Rapid fluorescence recovery after photobleaching confirms that bundles of cortical microtubules are not stable but exhibit properties consistent with dynamic instability. Dynamic microtubules offer a mechanism for rapid growth responses to a range of physiological stimuli.     

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.     

Modulation of melatonin receptors and G-protein function by microtubules

Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin-induced outgrowths in MT1-expressing CHO cells, also prevents MT1 receptor desensitization by affecting G(alpha)-GTP exchange on G-proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin-induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin-treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G-proteins. Depolymerization of microtubules during melatonin-induced desensitization, attenuated forskolin-induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of G(i alpha) protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into G(i) proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/G(q) coupling nor did it affect melatonin-induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin-induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on G(i) proteins and impact on downstream signaling cascades.     

Microtubule-disrupting agents affect two different events regulating the initiation of DNA synthesis in Swiss 3T3 cells.

The stimulatory effect of epidermal growth factor, alone or with insulin, on the rate of initiation of DNA synthesis in Swiss 3T3 cells can be synergistically enhanced by the addition of either Colcemid or colchicine at 1 microM. However, both Colcemid and colchicine can exert the synergistic effect only when added earlier than 8 hr of the prereplicative period (lag phase). Removal of Colcemid (which allows for rapid reassembly of microtubules) earlier than 10 hr of the lag phase results in a loss of the synergistic effect. This suggests that microtubules must remain disrupted for longer times to accomplish some putative event(s) necessary for increasing the rate of initiation of DNA synthesis. Preincubation of quiescent cells with either Colcemid or colchicine for 8 hr prior to adding epidermal growth factor, alone or with insulin, shortens the lag phase by about 4 hr, irrespective of the resulting rate of initiation of DNA synthesis. These results suggest that the state of microtubules is affecting independently at least two different events involved in regulating time initiation of DNA synthesis.     

Helical microtubule arrays in a collection of twisting tubulin mutants of Arabidopsis thaliana

Anisotropic expansion of plant cells requires organized arrays of cortical microtubules. Mutations in microtubule-associated proteins and a particular mutation in alpha-tubulins were reported to cause abnormal microtubule arrays and result in helical growth in Arabidopsis thaliana. However, the way in which these mutations affect the organization of microtubules remains unknown. We here identified 32 Arabidopsis twisting mutants that have either missense or amino acid deletion mutations in alpha- or beta-tubulins. Mutations were mapped to the GTPase-activating region in alpha-tubulin, intra- and interdimer interfaces of tubulin heterodimers, and lateral contact regions among adjacent protofilaments. These dominant-negative tubulin mutants were incorporated into the microtubule polymer and formed shallow helical arrays of distinct handedness along the long axis of the root epidermal cells. A striking correlation exists between the direction in which cortical helical arrays are skewed and the growth direction of elongating roots. The GTPase-activating-region mutant had left-handed helical arrays composed of highly stabilized microtubules, which could be decorated along the entire microtubule lattices with the otherwise tip-localized End Binding 1 protein. A mutation at the intradimer interface, on the other hand, generated highly dynamic microtubules and right-handed helical arrays. Cortical microtubules in wild type and these two tubulin mutants were composed mainly of 13 protofilaments. This comprehensive analysis of tubulin mutations provides insights into the mechanism by which tubulin structures influence microtubule dynamics and organization.     

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence

Abstract:  MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 p m melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of β-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.     

Involvement of the central nervous system in neuroendocrine mediation of osmotic and ionic regulation in the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

The presence of putative neurofactors within the central nervous system, i.e., the eyestalks (ES), ventral nerve cord (VNC), and supra-esophageal (SEG) and thoracic ganglia (TG), which are involved in osmotic and ionic regulation, was investigated in the euryhaline, freshwater shrimp, Macrobrachium olfersii. Homogenates were prepared from shrimps exposed for 6 hr to a high salinity medium (HSM, 21/1000 S) and were injected into shrimps subsequently maintained for 1, 3, or 6 hr in freshwater (FW, 0/1000 S) or HSM. Osmolality and sodium, chloride, and calcium concentrations were determined in single hemolymph samples removed at each time interval. Heart rates and wet weights were measured before and after experimental treatments. Exposure to HSM increased [Na+] and [Cl-] and heart rate. Injection of ES homogenate increased osmolality, [Na+] and [Cl-], and wet weight in shrimps maintained in FW; VNC homogenate also increased hemolymph [Cl-] in shrimps maintained in FW after injection, but reduced heart rate in shrimps subsequently exposed to HSM. Injection of TG homogenate reduced heart rate to a lesser extent in shrimps maintained in FW. Hemolymph [Ca2+] was not altered by homogenate injection. The exposure period of 6 hr to HSM appears to result in the accumulation of factors within the central nervous system that regulate the osmotic and ionic concentrations of the hemolymph, in addition to exerting antidiuretic and cardio-depressor actions. The coordinated action of these factors is intimately involved in the hyporegulatory processes that permit the survival of M. olfersii in media of elevated salinity.     

Cloning, expression, and properties of the microtubule-stabilizing protein STOP.

Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.     

The role of the microtubular system in LH/hCG receptor downregulation in rat Leydig cells

The mechanism by which hCG-induced LH/hCG receptor 'downregulation' occurs in rat Leydig cells is unknown. To study the role of microtubules in this process we used the microtubule inhibitors vinblastine and colchicine. 200 IU hCG causes a 92-95% decrease in [125I]hCG binding to testis homogenates within 6 h after injection. Both vinblastine and colchicine prevent this loss of hCG binding. Vinblastine or colchicine did not depress the raised serum testosterone levels following hCG injection, suggesting that these agents were not having a toxic effect on the Leydig cells or on the circulation to the testis such that access of the injected hCG was impeded. The morphological observation of vinblastine-induced bundles of filamentous material in Leydig cells is evidence of a direct action on the microtubular-microfilamentous system. This data strongly implicates a role for microtubules in the process of LH/hCG receptor downregulation that occurs in the Leydig cell following large injections of hCG.     

2',3'-Cyclic nucleotide 3'-phosphodiesterase: a membrane-bound, microtubule-associated protein and membrane anchor for tubulin

2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP) is firmly associated with tubulin from brain tissue and FRTL-5 thyroid cells as demonstrated by copolymerization with microtubules through several warm/cold cycles, the presence of CNP activity in purified tubulin preparations, and identical behavior during various extraction procedures. CNP acts as a microtubule-associated protein in promoting microtubule assembly at low mole ratios. This activity resides in the C terminus of the enzyme, which, by itself, promotes microtubule assembly at higher mole ratios. Phosphorylation of CNP interferes with its assembly-promoting activity, as does deletion of the C terminus, which leads to abnormal microtubule distribution in the cell. Submembranous colocalization of the proteins and CNP-dependent microtubule organization suggest that CNP is a membrane-bound microtubule-associated protein that can link tubulin to membranes and may regulate cytoplasmic microtubule distribution.     

Directed elongation model for microtubule GTP hydrolysis.

We propose a role for GTP hydrolysis in microtubule assembly in which the GTPase reaction serves to stabilize tubulin subunits in the microtubule. The GTPase reaction in tubulin subunits containing GTP at microtubule ends is presumed to occur predominately in subunits at one of the interfaces between a cap of GTP-containing tubulin subunit and a core of GDP-containing tubulin subunit in the microtubule, resulting in elongation of the core. The proposed model interprets the effects of GDP on microtubule assembly, using a reaction scheme in which GDP-containing tubulin subunits are able to add to microtubule ends. The model can account for the GTP requirement for microtubule assembly, the GDP inhibition of the rate for microtubule elongation, and the fact that a metastable state exists after the enzymic conversion of GTP to GDP, with microtubules which are at steady state. To account for the fact that the microtubule assembly and disassembly rates are nonlinearly dependent upon the tubulin subunit concentration and for the effects of GDP-containing tubulin subunits on the kinetic properties of microtubules, our scheme includes nonproductive as well as productive binding of GTP- and GDP-containing tubulin subunits. We compare our model with an alternative scheme [Hill, T. L. & Carlier, M. F. (1983) Proc. Natl. Acad. Sci. USA 80, 7234-7238], which interprets the effects of GDP on microtubule assembly using a reaction scheme in which GDP is able to exchange with GTP in GTP-containing tubulin subunits in the microtubule and in which the principal GTPase occurs in GTP-containing tubulin subunits at the microtubule/solution interface.     

Incorporation of Paramecium axonemal tubulin into higher plant cells reveals functional sites of microtubule assembly

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubule-organizing centers, which were revealed by the intense incorporation of exogenous tubulin, are discussed. These data shed light on the present debate on higher plant microtubule organizing centers.     

Radioautographic study of binding and internalization of corticotropin-releasing factor by rat anterior pituitary corticotrophs

In order to identify the anterior pituitary cell type(s) containing corticotropin-releasing factor (CRF) receptors and to study the internalization processes of this peptide by the target cells, radioautography was performed on rat anterior pituitaries removed at specific intervals (2-60 min) after intracarotid injection of [125I]iodo-CRF into intact and adrenalectomized female rats. In intact animals, all corticotrophs were labeled, whereas in the adrenalectomized animals about 80% of the hypertrophied corticotrophs (adrenalectomy cells) were. In control animals injected with both iodinated CRF and an excess of unlabeled peptide, no specific reaction could be detected. The time-course study in intact animals showed that 2 min after injection most silver grains were found over or within 160 nm of the plasma membrane. At the 5-min time intervals, grains were observed both over the plasma membrane and within the cytoplasm, associated with lysosomes, and the Golgi apparatus. Fifteen minutes after injection, grains were mostly found over lysosomes and the Golgi apparatus, whereas at the longest time intervals (30 and 60 min) almost no labeling could be detected. The results obtained in this study indicate that in the anterior pituitary CRF receptors are restricted to corticotrophs (as identified by electron microscopy) and that, after binding to the plasma membrane, CRF is rapidly internalized to Golgi elements and lysosomes.     

Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane.

A structure which appears to organize cytoplasmic microtubules in interphase mouse 3T3 cells can be visualized by immunofluorescence microscopy. Purified monospecific antibody against homogeneous tubulin from brain visualizes, in addition to cytoplasmic microtubules, a cytoplasmic polar structure as the focal point from which the microtubules seem to radiate. The structure is preserved after treatments that depolymerize cytoplasmic microtubules, i.e., exposure of cells to mitotic drugs or to low temperature. When cells recover from these treatments one end of each microtubule organizing structure acts as a nucleating center from which cytoplasmic microtubules grow toward the plasma membrane. Thus cytoplasmic microtubules assemble in vivo in an ordered unidirectional manner, and therefore the cell must be able to avoid the assembly of unwanted, unoriented, and disconnected microtubules. These results suggest that the assembly of tubulin into microtubules is regulated in vivo.     

Distribution and specific binding "in vivo" of iodinated growth hormones in the turtle Chrysemys dorbigni.

Bovine (bGH) and human growth hormone (hGH) labeled with ¹²⁵I were injected into turtle Chrysemys dorbigni, in order to study their distributions in tissues. The radioactivity was basically concentrated by the liver and kidney, reaching a maximum 4 hr after the labeled hormone injection. Only the liver showed a significant reduction of radioactivity uptake, when labeled growth hormone was injected together with an excess of unlabeled hormone. This reduction was dose dependent. Injection of [¹²⁵I]iodo-hGH produced higher uptake of radioactivity by the liver than [¹²⁵I]iodo-bGH. The study performed suggests: (1) specific uptake of hGH or bGH by the liver; and (2) the presence in the liver of both somatogenic and lactogenic binding sites.