Effect of light and darkness on pineal hydroxyindole-O-methyl transferase (HIOMT) in Japanese quail

The optimal incubation temperature for assay of the melatonin-forming enzyme, hydroxyindole-O-methyl transferase (HIOMT), of the Japanese quail pineal, was determined to be 47° C. In adult quail, HIOMT activity was high in continuous light and in the light phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$, and was low in continuous darkness and in the dark phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$. Age of the bird and length of the lighting treatment seemed to be important factors in demonstrating this effect of light and darkness on HIOMT activity.     

Effect of monensin and suckling on the GnRH induced luteinizing hormone surge and the effect of monensin on the postpartum interval in Brangus cows

Twenty-seven fall calving Brangus cows were randomly allotted to one of four treatment groups: nonsuckled monensin (NSM), suckled monensin (SM), nonsuckled control (NSC), and suckled control (SC). Cows were group fed 1.82 kg/hd/day concentrate and Coastal bermuda grass hay $\text{ad}$$\text{libitum}$. Monensin cows received 200 mg monensin/hd/day in the concentrate. At 0800 hr on day 21 postcalving, the calves were separated from the cows. Suckled monensin and SC cows were allowed to suckle their calves for 30 min at 6-hr intervals. Nonsuckled monensin and NSC cows were not suckled. Calves were given free access to the cows after 1400 hr on day 22 postpartum. At 0800 hr on day 22 postpartum, a blood sample was collected. A 100 μg GnRH challenge was administered IM at 0801 hr. Blood samples were collected at 15-min intervals for 6 hr postinjection. Changes in body weight and body condition from day 21 postpartum to the day of first estrus were not different (P>0.10) by dietary treatment. Monensin cows consumed 10.7% less hay than did the control cows. Serum luteinizing hormone (LH) following GnRH was greater (P<0.005) in suckled than nonsuckled cows. Control cows released more (P<0.005) LH in response to GnRH than did the monensin cows. The postpartum interval (to first estrus) for the monensin cows (92.4±14.7 days) was shorter (P<0.025) than the controls (138.5±9.5 days). A greater proportion (P<0.005) of the monensin cows (8 of 14) exhibited estrus by 90 days postpartum compared to the control cows (0 of 13). Monensin and suckling appear to exert independent and agonistic influences on pituitary function in the postpartum beef cow.     

Sulfation of gastrin in Zollinger-Ellison sera: evidence for association between sulfation and proteolytic processing

Sulfated gastrins were quantitated in sera from 15 patients with the Zollinger-Ellison syndrome (ZES) by specific radioimmunoassays. The total concentration of gastrin varied from 174 to 285000 pmol/1. Sulfated gastrins constituted $\text{44.8±5.5\%}$ (mean ± S.E.M.) of the gastrins in ZES sera compared with $\text{37.7±1.9\%}$ in sera from 100 control subjects ($\text{P>0.1}$). There was no correlation between gastrin concentration and sulfation ($\text{r=0.40}$). Gel and ion-exchange chromatography showed that up to 90% of the gastrins could be in the sulfated form. The highest degree of sulfation was found in sera where the small gastrin components dominated. Thus, the percentage of small gastrins (G-17 and G-14) correlated with the degree of sulfation ($\text{N = 15}$, $\text{r=0.75}$, $\text{P<0.01}$). We suggest therefore that proteolytic processing of the gastrin precursor and sulfation of tyrosyl are associated.     

An ultrastructural examination of everted rat jejunum

Male, albino, Sprague-Dawley rats were sacrificed by cervical separation. Segments of jejunum were excised, everted and examined with the electron microscope. Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed $\text{in}$$\text{situ}$ and $\text{in}$$\text{vitro}$: 1) an increase in the length of microvilli from (mean ± S. E.) 0.991 ± 0.011μ for normal tissue to 1.389 ± 0.023μ for everted tissue, 2) an increase in width of microvilli from (mean ± S. E.) 0.089 ± 0.001μ for normal tissue to 0.097 ± 0.001μ for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular “lakes” in the lateral spaces. The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates $\text{in}$$\text{vitro}$ as compared to $\text{in}$$\text{vivo}$.     

Circulating immunoreactivities of gastrin,glucagon and VIP after jejuno-ileal bypass

Seven Sprague-Dawley rats (404–440 g) underwent a 90% jejuno-ileal bypass (JIB); the functional loop consisted of $\text{1}\text{3}$ ileum and $\text{2}\text{3}$ jejunum with the bypassed loop being anastamosed to the ascending colon. Seven control rats were sham-operated. After 35 days, the rats were fasted 18 hours and venous blood was collected. Immunoreactivity of gastrin, measured with an antibody binding equally to G17 and G34, was higher in the plasma of the JIB (256±55 SEM pg/ml) than control (85±9 pg/ml) rats. This agrees with recent human studies but is in conflict with results in less mature rats. VIP levels were not significantly different. Glucagon-like immunoreactivity measured with antibodies specific for the C- and N-terminal regions of the hormone, respectively, were also higher in the JIB (510±40 and 129±15 pg/ml) rats.     

Evidence for central neuropeptidergic control of rumination in sheep

Effects of intracerebroventricular (ICV) vs. intravenous (IV) administration of tetragastrin, pentagastrin, CCK₈ and gastrin 17 on rumination were investigated in conscious sheep. Administered at 26 pmoles/kg ICV both tetra and pentagastrin induced a premature short (15–27 min) period of rumination only $\text{24±7}$ and $\text{23±9}$ min after food distribution in place of $\text{112±44}$ min in controls. Similar but less pronounced effects were observed for ICV administration of an equimolar dose of gastrin 17 whereas CCK₈ did not promote an early peciod of rumination despite its anorectic effects. Administered intravenously tetra and pentagastrin but not gastrin 17 caused early rumination only for 10 times higher doses. It is concluded that gastrin 17 and its C-terminal tetrapeptide may play a physiological role in the central control of rumination in sheep.     

Uptake and effects of ovarian steroids in the early pig embryo: In vitro and in vivo studies

The role of ovarian steroids in the preimplantation pig embryo was studied in vivo and in vitro. Twenty gilts were treated three times daily on days 1 to 4 after insemination with either 25, 100, 250, or 1000 mg progesterone in oil, and 17 gilts were injected with corresponding amounts of sesame oil (controls). All gilts were slaughtered 5 days after insemination and the embryos were recovered. Oviduct and plasma progesterone content were significantly (P<0.001) higher in gilts treated with 750 mg of exogenous progesterone per day. After 750 mg progesterone, oviduct progesterone content was twice as high as control levels, while after 3000 mg progesterone per day the levels in oviduct and uterus exceeded those of controls by five and seven times, respectively. In gilts treated with 750 mg progesterone per day, plasma progesterone levels were 177.4 ± 22.1 ng/ml ($\text{x}\text{±}\text{SD}$) on day 3 and 186.4 ± 69.2 ng/ml on day 5 and resembled values found in superovulated pigs with more than 40 ovulations. Excessive plasma progesterone values of 1014.6 ± 840.4 ng/ml on day 3 and 473.2 ± 197.2 ng/ml on day 5 were found after treatment with 3000 mg of progesterone per day. Treatment with up to 750 mg of exogenous progesterone per day, did not affect embryonic development, but 3000 mg per day resulted in a significantly (P<0.001) higher percentage of retarded and degenerate embryos compared to controls (71.8% versus 3.2%).In addition, the amount and specificity of uptake of ³H-labelled progesterone and estradiol-17 beta by pig blastocysts recovered from superovulated gilts were investigated after 6 hrs in vitro culture. The uptake of ³H-progesterone was 131.9 ± 56.9 counts per million (cpm) per 10 blastocysts, corresponding to 1.1 fmoles progesterone. The uptake was non-specific for it was only slightly reduced in the presence of a 100-fold excess of unlabelled progesterone (20.1%) or estradiol-17 beta (27.0%). The uptake of ³H-estradiol-17 beta was 133.9 ± 74.12 cpm per 10 blastocysts, corresponding to 1.3 fmoles estradiol-17 beta. The uptake was significantly (P<0.01) reduced by 67.7% in the presence of a 100-fold excess of unlabelled estradiol-17 beta. Apparent specific binding was 0.87 fmoles estradiol-17 beta per 10 blastocysts or 72.5 fmoles estradiol-17 beta per mg protein. The uptake was only slightly reduced in the presence of a 100-fold excess of unlabelled progesterone (23.3%). This significant inhibition could be determined after 2 hrs in vitro culture. There was no competitive inhibition after 20 min. of culture.Uptake by unfertilized ova and degenerate embryos recovered on day 5 was significantly smaller (51.8 ± 10.3 cpm per 10 eggs; P<0.001) than by blastocysts recovered on the same day. No competitive inhibition could then be determined. In vivo, preimplantation pig embryos seem to be rather insensitive to high progesterone levels. Excessive amounts of progesterone probably can be metabolized to a great extent. Progesterone seems to be taken up rather non-specifically by the pig embryo. The uptake and binding of estradiol-17 beta seems to be more specific. Studies are in progress to investigate the physiological role of estradiol-17 beta uptake in early embryonic development.     

Elevation of plasma tyrosine after a single oral dose of L-tyrosine.

Plasma tyrosine concentrations in twelve normal, fasting human subjects were significantly elevated 2–8 hours after they ingested 100 mg/kg or 150 mg/kg tyrosine. Mean plasma tyrosine levels were maximal after 2 hours, rising from $\text{69 ± 3.9}\text{to}\text{154 ± 9.5}\text{nmols/ml}\text{(}\text{X}\text{±}\text{SEM}\text{)}$ after the 100 mg/kg dose and to 203 ± 31.5 nmols/ml after the 150 mg/kg dose (p ≤ 0.001 for both doses). The mean tyrosine ratio (defined as the ratio of plasma tyrosine concentration to the sum of the concentrations of six other neutral amino acids that compete for the same blood-brain barrier uptake system) increased from $\text{0.10 ± 0.02}\text{to}\text{0.28 ± 0.04 (}\text{X}\text{±}\text{SEM}\text{)}$ 2 hours after the 100 mg/kg dose (p ≤ 0.001) and to 0.35 ± 0.05 2 hours after the 150 mg/kg dose (p ≤ 0.005). No side effects of orally-administered L-tyrosine were noted.     

The synthesis,characterization, and preliminary biological evaluation of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-L-1,2-dipalmitin

Recently, 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{DL}$-1,2-dipalmitin (VIa) was reported to inhibit the growth of L51784 cells in mice and of human colon carcinoma HCT-15 cells, also in mice. This paper describes the synthesis of a single diastereomer by conversion of 1-β-$\text{D}$-arabinofuranosylcytosine 5′-monophosphate (II) to the nucleoside 5′-phosphomorpholidate (III), followed by reaction with $\text{L}$-α-dipalmitoylphosphatidic acid (IV) to give 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{L}$-1,2-dipalmitin (V) in good yield. The separation of the product is described and its characterization by chromatography, elemental analysis, and spectroscopic methods. The lipophilic nature of V renders it insoluble in aqueous media and a method of sample preparation utilizing sonication techniques is described which provides a clear solution suitable for biological evaluation. In addition, the ability of V to inhibit the $\text{in}\text{vitro}$ growth of L1210 cells and of mouse myeloma MPC 11 cells is desscribed and compared with 1-β-$\text{D}$-arabinofuranosylcytosine (I) and other lipophilic prodrugs of I.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Theory of biological similarity revisited

A unified theory of biological similarity is proposed, based on dimensional analysis (mass M, length L, diameter D, time T) and on three postulates: (1) the constancy of body density in terrestrial mammals; (2) the elastic similarity criterion (Rashevsky and McMahon) where $\text{L ∝ D}{}^{\mathrm{<mtext>2</mtext><mtext>3</mtext>}}$; and (3) the proportionality between length (L) and time (T), which is valid for relaxation oscillators. The postulated theoretical model provides a satisfactory correlation (r = 0·9937) between the predicted reduced exponent (b) and 96 allometric exponents (b) obtained from experimental data concerning a number of morphologic and physiologic parameters in animals of different size.The reformulation of a theory of biological similarity is posited mainly for the “internal” organization of organisms, whereas a “mechanical” similarity should be applied when inertial forces are present during animal locomotion (kinematics).Since biological rhythmicity is based on relaxation oscillators (T ∝ L), while in “mechanical” similarity the pendulum ($\text{T ∝ L}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) is the paradigm of a self-sustained oscillation, these two are the limits of a continuous spectrum of similarity criteria, where the exponent of the time dimension (T) is the essential factor.The four-dimensional nature of biological space is discussed (W ∝ L⁴), and due to the postulated isometry of length (L) and time (T), periodic phenomena conform to $\text{T ∝ W}{}^{\mathrm{<mtext>1</mtext><mtext>4</mtext>}}$.     

Reproductive studies of Brahman cattle I. Behavioral effect of various dose levels of Estradiol-17β upon ovariectomized Brahman,Brahman × Hereford and Hereford cows

Six ovariectomized mature cows each of Brahman (B), Brahman × Hereford (BH) or Hereford (H) breeding were injected intramuscularly with Estradiol-17β (E). Dose levels of 1, 2, 4 and 8 mg E were given in 2 ml corn oil. Cows were allowed a 2 week recovery period between treatments. After injection the cows were placed with 18 epididymectomized bulls and observed constantly for 36 hours. B failed to accept the bulls at any E dose level. Proportions of BH accepting the bull were $\text{2}\text{6}$, $\text{6}\text{6}$, $\text{6}\text{6}$ and $\text{6}\text{6}$ and proportions of H accepting the bull were $\text{5}\text{6}$, $\text{6}\text{6}$, $\text{5}\text{6}$ and $\text{6}\text{6}$ at 1, 2, 4 and 8 mg E, respectively. BH were less responsive at 1 mg E than H (P<.10) and B were less responsive at any level (P<.005). The number of stances increased significantly with dose level (P<.005) and a breed × dose level interaction (P<.10) was found. The duration of standing estrus behavior was longer in H cows at 1 and 2 mg E than in BH (P<.05) but was identical at 4 and 8 mg E. Duration of estrus was shorter in B except at the 2 mg dose level. Breed (P<.005) and breed × dose level interactions (P<.05) were found. Response time (injection of E to stance event) did not differ between dosages of E within breed groups. However, response time was significantly longer in B (19.3 hrs, P<.05) versus the response time of either H (10.1 hrs) or BH (12.8 hrs). If homosexual stance behavior is accepted as estrus, B were less responsive at 1, 2, 4 and 8 mg E than were BH or H (P<.10).     

Pregnancy diagnosis in the domestic horse through direct urinary estrone conjugate analysis

Urinary estrone conjugates were measured directly by radioimmunoassay (RIA) in 20 pregnancies from preconception diestrus to day 78 of pregnancy. High performance liquid chromatography separation defined estrone sulfate (ES) as the predominant immunoreactive peak which accounted for 94% to 97% of the total immunoreactivity after chromatography. Diestrous values indexed by creatinine were 0.15 ± 0.07 micrograms/mg Cr, $\text{x}\text{± SEM}$ as compared to estrous values which rose to 0.47 ± 0.14 micrograms/mg Cr, $\text{x}\text{± SEM}$. Urinary ES concentrations significantly increased (P = 0.0001 in pregnant mares from day 35 to day 47 (1.21 ± 0.12 micrograms/mg Cr) as compared to day 25 to day 34 (0.27 ± 0.01 micrograms/mg Cr). Measurement of urinary ES may provide an alternate or augmentive method of pregnancy diagnosis in the domestic mare.     

Daily sperm production of zebus (Bosindicus) estimated by quantitative histology of the testis

Microscopic parameters were studied quantitatively in testes from six Nelore zebus ($\text{Bos}$$\text{indicus}$), aged 4 to 6 years, with normal spermatogenesis, which were kept at sexual rest. Ratios of germ cell nuclei, counted in cross sections of seminiferous tubules, indicated that cellular losses in the spermatogenic process of the zebu are higher than those observed in taurines. Daily sperm production, estimated from the number of round spermatids in stage 1 of the cycle of the seminiferous epithelium and the duration of this cycle, was (mean ± SD) 12.2 ± 0.9 × 10⁶ spermatids/gram of testis parenchyma/day and 2.6 ± 0.5 × 10⁹ spermatids/testis/day. These values are smaller than those of taurines.     

Circadian control of singing in crickets: Two different pacemakers for early-evening and before-dawn activity

(1) Under LD 12:12 and constant temperature, calling song patterns of the Australian Field Cricket Teleogryllus commodus were frequently divided into two more or less distinct activity components. The first began 1–3 h before the $\text{L}\text{D}\text{-transition}$ and ended with the sudden lights-off, whereas the second was restricted mainly to the second half of the night. In subsequent continuous light (LL) the circadian activity pattern was usually a unimodal band. Its onset slope was always a continuation of the first onsets in the preceding LD-cycle. In those cases where shortly after the $\text{LD}\text{LL}$ transition the rhythm split into two components, both corresponded to those in LD. (2) In “atypical” LD-aatterns only the second component appeared during the late night. In such cases, the onset slope of the subsequent free-running rhythm did not start at the preceding LD-entrained onsets, but was advanced by several hours. (3) In free-running rhythms the mean value of the onset slopes ($\text{τ}{}_{\mathrm{o}}\text{= 25.1 ± 0.07}\text{h}$) did not differ significantly from that of the end slopes ($\text{τ}{}_{\mathrm{e}}\text{= 25.0 ± 0.07}\text{h}$). However, in individual activity patterns, simultaneous differences in onset and end slopes of up to 0.7 h were found. τ_o- and τ_e-values recorded directly after the $\text{LD}\text{LL}\text{-transition}$ did not correlate with the preceding phase angle difference between the entrained activity and the zeitgeber. (4) Under constant conditions after-effects such as spontaneous period changes and phase shifts in the slopes of activity onset and/or end occurred mainly between five and 20 days after the $\text{LD}\text{LL}\text{-transition}$. (5) Period changes and phase delays in the onset and end slopes were also found when crickets were exposed to low temperature pulses (2 ± 2°C, 2h duration). Following the cold treatment both period changes and phase shifts in the onset slope frequently differed from those in the end slope. (6) The results are consistent with a concept of two activity components controlled by weakly coupled circadian pacemakers. This is clearly evident in split rhythms but is also true for unimodal patterns. Normally the two components partly overlap and their existence is concealed in the undivided activity band. They are only revealed in unimodal patterns if individual oscillatory properties such as different period lengths or phase shifts appear in the onset or the end slope.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.