Effects of ATP on calcium binding to synaptic plasma membrane

The release of labeled norepinephrine from preloaded synaptosomes requires the presence of potassium and calcium. ATP-dependent binding of calcium to synaptic plasma membranes (SPM) may provide a means of maintaining the cation in a readily available pool for the triggering of transmitter release. A high Ca-binding capacity was demonstrated in SPM. The Km for calcium is 5.5 X 10(-5) M. The dependence of the system on the gamma phosphate of ATP was demonstrated by an increase in Ca-binding with increasing ATP concentration and by competitive inhibition of binding by ADP and AMP. Magnesium is also required for ATP-dependent Ca-binding. The optimum pH for the Ca binding was 7.0. Pretreatment of SPM with phospholipase A2 lowered the binding capacity. Sulfhydryl groups are also critical for ATP-dependent Ca binding to occur. A model for ATP-dependent Ca-binding was proposed.     

Regulatory effects of ATP and calcium on the myofibrillar ATPase of frog sartorius muscle

The ATPase activity of frog sartorius myofibrils has been studied at 1.5°C using different concentrations of ATP and calcium. The progressive activation of the ATPase activity at Ca-concentrations between pCa 8 and pCa 4 is paralleled by increases in Ca-binding. Similar to the findings of Weber and Bremel (1972) on rabbit psoas myofibrils more calcium is bound at pCa 5 – 7 in presence of 10 μM ATP than at 2 mM ATP. The observation, that in presence of 2 mμM N-ethyl maleimide/mg myofibrillar protein Ca-binding is essentially abolished at the lower calcium levels and becomes reduced by 30 – 40% at pCa 4 – 6, has been explained in terms of a Ca-binding site on the myosin. Using carbon-14-labelled ATP it could be demonstrated that the lower ATPase activity at pCa 7 or pCa 9 is associated with an increase in nucleotide binding, which is much reduced at a pCa of 4. However, removal of calcium from the medium does not increase the number of nucleotide binding sites as has been reported for rabbit myofibrils. A kinetic interpretation of the ATPase and ligand binding studies is offered.     

Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized, and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion

Opiate peptides are thought to modulate the pattern of LH release in female rats. We tested the hypothesis that changes in proopiomelanocortin (POMC) gene expression occur in proestrous (PRO) and ovariectomized (OVX) steroid-treated rats which may explain their unique patterns of LH secretion. Using in situ hybridization, we examined whether diurnal changes in POMC gene expression occur in the arcuate nucleus. Four groups of rats were used in this study. 1) PRO rats were used after exhibiting at least two consecutive 4-day estrous cycles; 2) OVX rats were killed 9 days after ovariectomy; 3) estradiol (E2)-treated rats were OVX for 7 days and then treated for 2 days; and 4) E2-progesterone (P4)-treated rats were treated with E2 as described above, and on day 9 at 1030 h, P4 was administered. Rats were killed at 2300, 0300, 1000, 1300, 1500, 1800, or 2300 h, beginning on the evening of diestrous day 2 or day 8 after ovariectomy. POMC gene expression exhibited a diurnal rhythm on PRO. Levels of mRNA rose during the morning, peaked between 0300-1000 h, and decreased by 2300 h. In E2-treated rats, which exhibited a LH surge similar in timing to the PRO surge, POMC mRNA levels exhibited a diurnal rhythm strikingly similar to that observed in PRO animals. OVX abolished the rhythm; however, average POMC mRNA levels across the 24-h period were not significantly different from those in PRO or E2-treated rats. P4 treatment increased POMC mRNA levels by 2300 h compared to those in all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estradiol-17 beta and estriol on their binding sites in the rabbit uterus

1. Receptors for estradiol-17 beta (E2) and estriol (E3) were detected in the rabbit uterus. 2. Saturation analysis of estrogen binding sites in the cytosol showed that the dissociation constants of E2 and E3 for the high affinity binding sites were 1.8 +/- 0.5 nM and 2.3 +/- 0.3 nM, respectively, when dextran-coated charcoal was used to isolate free and bound ligands. 3. To eliminate non-specific (cross) bindings to their receptors, effects of unlabeled E2 and E3 on [3H]E3 and [3H]E2 bindings was examined. 4. [3H]E2 cytosol binding was observed to be specific for E2 and [3H]E3 cytosol binding was more specific for E3. 5. E2 priming to rabbits increased the binding sites for both E2 and E3, which was also more potent than E3 priming. 6. Moreover, the increase in E2 binding sites was greater than that in E3 binding sites. 7. These findings may suggest that there are separate binding sites for E2 and E3 in rabbit uterus and that synthesis of their binding sites is regulated by E2 but not E3.     

Dominant expression of porcine Calbindin-D9k in the uterus during a luteal phase

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue.     

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.     

Release of prostaglandins and leukotrienes from the rat uterus is an early estrogenic response

The early estrogenic responses are considered to be involved in inducing embryo implantation in a progesterone (P4)-primed uterus. Because of their involvement in the process of implantation and decidualization, prostaglandins (PGs) and leukotrienes (LTs) could be the mediators of early estrogenic responses in a P4-primed uterus. Therefore, temporal effects of estrogen on the production and/or release of PGF2, PGF2 alpha, LTB4 and LTC4 by the P4-primed uterus of hypophysectomized rats were examined. Hypophysectomized mature female rats were injected for 4 days with P4 (2 mg/rat, s.c.) or with P4 plus a single injection of estradiol-17 beta (E2) (100 ng or 200 ng/rat, i.v.) on the last day of P4 treatment. In one set of experiments, animals were killed at 0.5, 2, 4, 8, 12 and 30th after the last steroid treatment. The production of PGs and Lts by uterine homogenates was measured by radioimmunoassays (RIAs). The production of PGE2 and PGF2 alpha in P4-treated animals showed peaks at 2, 6 and 12h. The superimposition of E2 on P4 treatment induced a higher production rate of PGE2 and PGF2 alpha at 0.5h and abolished the peaks induced by P4 at 2h, but not the peaks at 6 or 12h. Irrespective of the kind of steroid hormonal treatments, uterine production of LTs showed a rapid decline between 6 and 8h followed by a sharp rise at 12h. The superimposition of E2 on P4-treatment again increased the production rates of LTB4 and LTC4 at early hours, i.e. at 0.5 and 2h, respectively, as compared to P4 treatment only.     

Effect of progestins, estradiol, and coenzymes NAD and NADPH on the interconversion of estradiol and estrone in rabbit uterus in vitro

The biotransformation of estradiol (E2) and estrone (E1) in the uterus of rabbits treated with norgestrel (NG), norethindrone (NET), norethindrone acetate (NETA), progesterone (P4), and E2 either by subcutaneous injection in oil or by intrauterine steroid-releasing silastic implants was carried out under an in vitro short-term incubation system. The studies have shown that E2 stimulates 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSD) much more than P4 as compared to untreated controls. The kinetic studies on E2 metabolism in the presence of added coenzyme NAD showed an initial rapid estrone formation and a gradual reconversion of E1 to E2. The addition of NADPH, ATP, and glucose-6-phosphate facilitates the reconversion of E1 to E2. The interconversion of E2 and estrone in the presence of coenzymes was five- to ten-fold higher in the endometrium than in the myometrium per milligram protein. Both E2 and progestins stimulate the uterine 17 beta-OHSD activity in rabbit uterus. This study further suggested that the hormone-induced metabolism of estradiol and estrone in the rabbit uterus is essentially modulated by the availability of coenzymes.     

Deciduomal response in androgenized rats: effect of age and steroid replacement therapy

Deciduomal response was studied in female rats androgenized with a single injection of 1 mg of testosterone propionate at 5 days of age. Endometrial scratching in immature rats (33 days) elicited a better response in androgenized rats (AF) than in controls (NF) following induction of ovulation or steroid replacement therapy. In adult females receiving cervical stimulation at estrus or induction of ovulation, strong deciduomal response was obtained in NF rats and no response was observed in AF rats. In ovariectomized (OVX) rats receiving 2 mg of progesterone (P), the response in AF was only 50% that of NF rats. Addition of 0.1 mg of estradiol (E2) enhanced the decidualization in NF rats but completely abolished that of AF rats. Following ovariectomy and a period of 12-15 days without any exogenous hormone, an E2 priming treatment (0.2 or 0.5 micrograms) for 3 days followed by a replacement therapy (2.0 mg P + 0.1 or 0.15 micrograms E2) allowed good response in NF rats. The response was reduced by 30-35% in AF rats receiving 0.1 micrograms of E2 during the replacement therapy and by 66% in AF rats receiving 0.15 micrograms of E2. These results indicate that in AF rats the reduction of the response is age dependent, the uterus is more sensitive to E2 than is the uterus of NF rats and the growth response is always submaximal.     

Effects of streptozotocin-induced diabetes mellitus on some bone turnover markers in the vertebrae of ovary-intact and ovariectomized adult rats.

Diabetes mellitus and estrogen deficit are known causes of osteopenia in animal models as well as in humans. In the present work, the combined effect of ovariectomy and diabetes was investigated. Diabetes was induced in ovary-intact and ovariectomized female Wistar rats with a single injection (50 mg/kg body weight, i.p.) of streptozotocin. The rats were administered insulin (I) daily or 17-beta estradiol (E2) on alternate days for a period of 35 days and sacrificed. Serum calcium (Ca2+), phosphorus (P), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), vertebral ALP, collagen, and glycosaminoglycans were estimated. The levels of serum Ca2+ and P increased in diabetic rats, but decreased after I or E2 treatments. Serum ALP and TRAP activity increased in the ovary-intact and ovariectomized diabetic rats. Vertebral ALP activity increased in ovariectomized diabetic rats, but decreased in diabetic rats, which were treated with I or E2. In the vertebrae, TRAP activity was elevated as a result of diabetes, but this was prevented by insulin or estradiol. Diabetes induced a decrease in total collagen in the vertebrae, while I or E2 treatment induced an increase. The levels of chondroitin sulphate and heparan sulphate decreased significantly in the vertebrae of both ovary-intact and ovariectomized diabetic rats, while hyaluronic acid increased. In conclusion, diabetes and ovariectomy each seem to affect the process of matrix formation and mineralization in the bone, and this is aggravated by the combination of diabetes and ovariectomy. The effects of I and E2 were similar, and both hormones reversed the changes brought about by diabetes.     

A molecular model of cooperative binding of Ca2+ with rhodopsin molecules in photoreceptor membranes]

Kinetics of calcium binding by photoreceptor membranes of cattle retina in concentration Ca2+ 0.5 and 1.0.10(-5) M in 5 mM tris-HCl buffer, pH 7.4 at 37 degrees C has been studied. Such kinetics is of oscillating nature. Analysis of calcium binding process curves by photoreceptor membranes allow to conclude, that crystalline areas of rhodopsin (receptor domains) can be formed in the structure of photoreceptor membranes. Conformation states and structure of rhodopsin molecules Ca-binding sites in receptor domains depend on the presence of Ca2+ in the medium. The structure of rhodopsin molecules Ca-binding sites in receptor domain formed in the presence of Ca2+ in the medium was proposed. According to the Hodgkin and Huxley conception concerning the properties of Na(+)- and K(+)-channels, the receptor domain with such a structure of rhodopsin molecules Ca-binding sites can represent the conjugate system of Na(+)- and K(+)-channels. Molecular mechanisms of photoreceptor and nerve cells excitation was also proposed.     

Biology and physiology of Calbindin-D9k in female reproductive tissues: Involvement of steroids and endocrine disruptors

Although Calbindin-D9k (CaBP-9k), a cytosolic calcium binding protein which has calcium binding sites, is expressed in various tissues, i.e., intestine, uterus, and placenta, potential roles of this gene and its protein are not clearly understood. Uterine CaBP-9k may be involved in controlling myometrial activity related with intracellular calcium level and is not under the control of vitamin D despite the presence of vitamin D receptors. But, it is under the control of the sex steroid hormones, estrogen (E2) and progesterone (P4), in female reproductive systems including the uterus and placenta. Thus, in this review, we summarize recent research literature in regards to the expression and regulation of CaBP-9k in mammals and introduce the research data of recent studies by us and others.     

Kinetics of glucocorticoid interaction with wild-type and variant receptors

Data concerning the short- and longterm effects of ovariectomy on the levels of estrogen binding sites in the rat uterus and liver are presented. The information increases the understanding of the regulation of estrogen receptor synthesis. The circulating estrogen level is suggested to affect receptor synthesis in the uterus and liver differently. Shortly after gonadectomy (2–20h), an elevation in the concentration of cytoplasmic binding sites in the uterus of 35% was observed, whereas no effect was seen in the liver cell. A longer period of time after ovariectomy (2–3 months) caused a reduction in the number of uterine receptor sites by 74%, whereas in the liver an increase of 84% was detected.     

Possibility of anti-estrogen action from estriol specific binding sites in rabbit uterus

1. This study was designed to investigate the clomiphene or tamoxifen binding to receptor sites for estradiol-17 beta (E2R) and estriol (E3R) in the rabbit uterus. 2. Those so-called anti-estrogenic compounds tended to inhibit E2-E2R and E3-E3R bindings equally. 3. The inhibitor constant of clomiphene for E2R was approximately 3.8 x 10(-8) M at 4 degrees C and that for E3R approximately 1.8 x 10(-8) M at 4 degrees C in a given case, determined by charcoal assay. 4. It is suggested that the anti-estrogenic compounds demonstrate their effects after binding either to E2R or to E3R. 5. There were some tissue differences of the contents between E2R and E3R. For example, the uterus and the cortex contained E2R, and the pituitary E3R more than the other.     

Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats

During the perinatal period, calcium metabolism is stressed. As intestinal Ca-binding protein is considered as a molecular expression of the hormonal effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), Ca-binding protin measurements may document the vitamin D roles during this period. We describe the variations of Ca-binding protein concentrations in the rat during the last 5 days of gestation, in the maternal duodenum, placentas, fetal membranes and fetal intestines. We also report intestinal Ca-binding protein changes from birth until weaning. The evolution of the maternal intestinal Ca-binding protein, which increases on day 19.5 of gestation, is consistent with that of calcium intestinal absorption and may be explained by increased 1,25(OH)2D3 production. Placental Ca-binding protein rises from day 17.5 until the end of gestation, and may be related to the profile of calcium transfer from mother to fetuses. It is noteworthy that the placental Ca-binding protein is predominantly found in the fetal part of the organ where materno-fetal exchanges occur. The yolk sac synthesizes substantial amounts of Ca-binding protein. In the fetal membranes, Ca-binding protein plateaus from day 17.5 until day 20.5 and decreases on day 21.5. The Ca-binding protein presence in the fetal placenta and in the yolk sac may suggest that these tissues are also targets for vitamin D. In the fetus the intestinal Ca-binding protein s is detected as early as day 17.5 of gestation and increases markedly during the last day of gestation. From birth and during the first 3 weeks of postnatal life, the intestinal Ca-binding protein concentration does not change. It undergoes a sharp rise just at the time of weaning. We have also shown that the specific distribution of Ca-binding protein along the intestine is acquired during intrauterine life and does not change with sucking or weaning. The two main changes of intestinal Ca-binding protein, observed just before birth and at weaning, may reflect the intestinal maturation and/or variations in vitamin D metabolism.     

Early treatment of young female rats with progesterone delays the aging-associated reproductive decline: a counteraction by estradiol

We have recently reported that successive treatments of young virgin rats with progesterone (P) implants produce elevated circulating P and consistently low estradiol (E2) concentrations, and subsequently delay the aging-associated reproductive decline. Inasmuch as E2 has been implicated in causing the loss of regular estrous cyclicity in aging rats, the present study examined if the concomitant presence of moderately increased circulating E2 levels could counteract the effects of P implants on reproductive aging. Starting at 3 1/2 mo and continuing to 8 mo of age, regularly cyclic, virgin rats received either s.c. Silastic implants of P (P-implanted), blank Silastic implants (virgin controls), or P + E2 implants (P + E2-implanted) for 3 wk, followed by implant removal for 1 wk. Each of these implant treatments was repeated in the same female rats 5 times. Blood samples were obtained on different days of the estrous cycle from the control group and on Day 11 of successive treatments with P or P + E2 implants for measurements of serum P and E2 values. At 8 1/2 and 10 mo of age, estrous cyclicity of these same virgin rats was again monitored, and 10-mo-old regularly cyclic females from each treatment group were mated with young fertile males to complete term pregnancies. While virgin controls showed cyclic increases in E2 and P secretion during the estrous cycle, P-implanted virgins exhibited consistently low serum E2 and moderately increased P levels during 5 successive treatments. The latter indicates a potent inhibition of ovarian E2 secretion by P implants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandin E1 or E2 (PGE1; PGE2) on luteal function and binding of luteinizing hormone in nonpregnant ewes

Two studies were conducted to determine the effects of PGE1 or PGE2 on luteal function and binding of luteinizing hormone (LH) to luteal cell membranes in nonpregnant ewes. In Study I, ewes (n=5 per group) received an injection of vehicle (VEH) or 333 micrograms of PGE1 or PGE2 into the tissue surrounding the ovarian vascular pedicle (intrapedicle) on day 7 postestrus. Systemic progesterone concentrations of PGE1-treated ewes were greater (P less than 0.01) than those of VEH-treated ewes at 24 and 48 hr after injection. For PGE2-treated ewes, progesterone concentrations were greater (P less than 0.01) than for VEH-treated ewes only at 24 hr. Neither PGE1 nor PGE2 affected luteal weights or LH binding capacity at 48 hr. Treatment with PGE1, however, increased (P less than 0.10) endogenously bound LH at this time. In Study II, ewes (n=5 per group) received an intrapedicle injection of VEH, or 10 mg of PGE1 or PGE2 on day 8 postestrus. Systemic progesterone concentrations in PGE1-treated ewes were less (P less than 0.01) than for VEH-treated ewes at 24 hr, but by 72 hr were not different from those of VEH-treated ewes. For PGE2-treated ewes, systemic progesterone declined steadily to reach low values by 72 hr. Prostaglandin E2 had no effect on luteal binding of LH at 72 hr, whereas PGE1 increased (P less than 0.05) LH binding capacity and endogenously bound LH. Although PGE2 had no apparent affect on luteal binding of LH in these studies, PGE1 may enhance the function of ovine corpora lutea by stimulating an increase in their binding of LH and capacity to bind LH when the CL receives a luteolytic signal.     

Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus

Progesterone (P4) is unequivocally required to maintain a uterine environment conducive to pregnancy. This study investigated the effects of P4 treatment on expression of selected growth factors (fibroblast growth factor 7 [FGF7], FGF10, hepatocyte growth factor [HGF], and insulin-like growth factors [IGF1 and IGF2]), their receptors (MET, FGFR2(IIIB), and IGF1R), and IGF binding proteins (IGFBPs) in the ovine uterus. Ewes received daily injections of corn oil vehicle (CO) or 25 mg of P4 in vehicle from 36 h after mating (Day 0) to hysterectomy on Day 9 or Day 12. Another group received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Day 8 through Day 12. Endometrial FGF10 mRNA levels increased between Day 9 and Day 12 and in response to P4 on Day 9 in CO-treated ewes, which had larger blastocysts, and were substantially reduced in P4+RU486-treated ewes, which had no blastocysts on Day 12. Endometrial FGF7 or HGF mRNA levels were not affected by day or reduced by RU486 treatment, but MET mRNA levels were higher in P4-treated ewes on Day 9 and Day 12. Levels of IGF1, IGF2, and IGF1R mRNA in the endometria were not affected by early P4 treatment. Although stromal IGFBPs were unaffected by P4, levels of IGFBP1 and IGFBP3 mRNA in uterine luminal epithelia were increased substantially between Day 9 and Day 12 of pregnancy in CO-treated ewes and on Day 9 in early P4-treated ewes. Therefore, FGF10, MET, IGFBP1, and IGFBP3 are P4-regulated factors within the endometrium of the ovine uterus that have potential effects on endometrial function and peri-implantation blastocyst growth and development.     

Interaction of pregna-D'-pentaranes--pentacyclic derivatives of progesterone--with gestagen-binding sites of uterine cytosol

The interaction of promegestone (R-5020), progesterone (P) and its derivatives having and additional carbocyclic D' (pregna-D'-pentrans) with progestin-binding cytosol system of the uterus was studied in different species (rabbits, rats, guinea-pigs and men). A comparative analysis of the competitive binding data for the mentioned compounds has shown interspecies differences in ligand specificity. Two types of binding sites for 3H-D'-pentran (in contrast to R-5020 and P) have been detected in rabbit uterus cytosol, both in intact and estrogenized animals. However, in rabbits, estrogenization altered the values of the apparent equilibrium constants and binding capacities. At the same time, the interaction of pentran with progestin-binding sites in guinea-pig and human uterus cytosol is nonspecific. It is suggested that the features of the interaction of 3H-D'-pentran with its binding sites in rabbit uterus cytosol may be determined by an increase in hydrophobic bond.