摘要:

AbstractThe effect of transforming growth factor‐α (TGFα) on granulosa cell differentiation, as assessed by the acquisition of aromatase activity, was evaluated in vitro by using a primary culture of rat granulosa cells. Harvested from immature, diethylstilbestrol‐treated rats, granulosa cells were cultured under serum‐free conditions for 72 hr in the presence of saturating concentrations (10−7M) of aromatase substrate androstenedione with or without the specified experimental agents. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen was negligible, remaining unaffected by treatment with TGFα; (10 ng/ml) by itself. Whereas treatment with follicle‐stimulating hormone (FSH) resulted in a substantial increase in the extent of aromatization, concurrent treatment with TGFα: (10 ng/ml) resulted in significant (P<0.05), yet reversible inhibition (78 ± 5.6%) of FSH action. Significantly, this effect of TGFα could not be accounted for by a decrease in cellular viability or plating efficiency nor by a decrease in the number of cells or their DNA content. Although independent of the FSH dose employed, the TGFα effect proved dose‐ and time‐dependent, with an apparent median inhibitory dose (EC50) of 0.33 ± 0.04 ng/ml, and a minimal time requirement of 48 hr. Capable of substantial inhibition of the forskolin‐stimulated accumulation of extracellular adenosine 3′, 5′ cyclic monophosphate (cAMP) and estrogen, TGFα had a measurable albeit limited effect on N6, 2‐′O‐Dibutyryladensine 3′:5′‐cyclic monophosphate‐supported estrogen production. Relative potency comparison revealed epidermal growth factor (EGF; EC50= 0.24 ± 0.03ng/ml) and TGFα to be virtually equipotent as regards the attenuation of FSH‐stimulated estrogen biosynthesis. Taken together, our findings indicate that TGFα, like EGF, acting at subnanomolar concentrations, is capable of attenuating the FSH‐stimulated (but not basal) accumulation of estrogen. This effect of TGFα proved time‐ and dose‐dependent, involving virtually complete neutralization of FSH action at site(s) both proximal and distal to cAMP generation. As such, these findings provide yet another example of the remarkable qualitative and quantitative similarities between EGF and TGFα, thereby reaffirming the prospect that ligands of the EGF/TGFα receptor may play a

ISSN:0730-2312    

DOI:10.1002/jcb.240330102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractVarious glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2‐ to 6‐fold by 10−7M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6‐bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin‐stimulated phosphorylation but to a smaller extent than that for phpsphofructokinase or phosphoglycerate mutase.The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate‐limiting step in glycolosis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 μM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the β‐subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10−9and 10−8M insulin), and cation requirement (Mn2+>Mg2+>>Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin‐stimulated increas

ISSN:0730-2312    

DOI:10.1002/jcb.240330103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractTo study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immuno‐globulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5′‐triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti‐receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase α by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating

ISSN:0730-2312    

DOI:10.1002/jcb.240330104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractThe cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio‐specific cleavages occur at glutamine‐glyeine (Q‐G) pairs that are recognized by the viral‐encoded proteinase 3C (formerly called P3–7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN‐III‐C3–7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino‐ and carboxy‐terminal Q‐G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN‐III‐C3–7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site‐specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy‐terminal Q‐G cleavage site. The mutant recombinant plasmid (designated pIN‐III‐C3‐μ10) directs the synthesis of a bacterial‐polio precursor polypeptide that is like the wild‐type construct (pIN‐III‐C3–7c). However, unlike the wild‐type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino‐terminal Q‐G site but not at the carboxy‐terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable su

ISSN:0730-2312    

DOI:10.1002/jcb.240330105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractAspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin. and chymosin). lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93–109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a “pro” enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens.It is the aim of this article to summarize the major models of structure‐function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic pr

ISSN:0730-2312    

DOI:10.1002/jcb.240330106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractHormones and growth factors are generally released from larger precursors by limited proteolysis. The causative agents remain poorly defined with respect to location and properties. One subset of proteases, the glandular kallikreins, have been implicated in a few cases, in part because of their specific association with mature forms of some hormones. However, limited distribution and low copy number in some species cast doubt on this hypothesis, and they may well play other physiological functions that remain to be elucidated.

ISSN:0730-2312    

DOI:10.1002/jcb.240330107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240330101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY