The development of carnitine synthesis from γ-butyrobetaine in the rat

The ability of the rat liver to synthesize camitine from γ-butyrobetaine increases from low values in the fetus to adult values on the 8th day after birth. The rate of synthesis of camitine is greater when determined in the high-speed supernatant than in the low-speed supernatant of the liver. No synthesis could be shown to occur in neonatal rat kidney or neonatal brown adipose tissue.     

The isolation and amino acid sequence of the β- and γ-subunits of the lectin from the seeds of Dioclea Grandiflora

Although the two smaller β- and γ- subunits of the lectin from Dioclea grandiflora were clearly resolved by sodium dodecyl sulphate (SDS) gel electrophoresis, the concensus of other techniques including ultracentrifugation, isoelectric focusing in 8 M urea, size-exclusion chromatography in dissociating solvents and amino acid and sequence analysis indicated that they were similar in molecular size and that they had arisen either by a single enzymic cleavage at Asn¹¹⁸-Ser¹¹⁹ in the middle of the 237 residue-long mature α-subunit or by multiple cleavages occurring during post-translational processing of intermediates. The existence of minor forms of the β- and γ- subunits resulting from a cleavage at Asn¹²⁴-Ser¹²⁵ of the α-subunit was also recognized. The results indicated that the apparent difference in molecular size of the β- and γ-subunits deduced from SDS-gel electrophoresis could be explained by the anomalous behaviour of both subunits in this separation technique. The structural features of the D. grandiflora lectin are compared with those of concanavalin A obtained from seeds of the botanically related Canavalia ensiformis.     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Immunohistochemical localization of γ-melanocyte-stimulating hormone in the rat pituitary gland

A third melatropin fragment named γ-MSH has been described in the N-terminal portion of the common precursor of bovine ACTH and β-LPH by Nakanishi et al. (Nakanishi, S., Inoue, A., Kita, K., Nakamura, M., Chang, A.C.Y., Cohen, S.N. and Numa, S., Nature, 278 (1979) 423–427). In order to determine if immunoreactive γ-MSH was present in the rat pituitary gland and to accurately localize this peptide, an immunocytochemical localization of γ-MSH was conducted at both light and electron microscopic levels. Specific immunostaining was detected in stellate cells scattered throughout the pars distalis and in all the cells of the pars intermedia. At the ultrastructural level, immunoreactive γ-MSH was only observed in the lipocorticotrophs. Using serial ultrathin sections, it was shown that the secretory granules which contain ACTH were also labeled for γ-MSH. These results suggest that fragment(s) of the common precursor of ACTH and β-LPH and/or the whole common precursor is released with peptides of known biological activity.     

The characterization of the whey proteins of guinea-pig milk: The isolation and properties of α-lactalbumin

1. The whey proteins of guinea-pig milk were examined by electrophoresis on paper, cellulose acetate, starch gel and polyacrylamide gel. 2. Two major proteins were detected, one of which was identified as blood serum albumin. 3. The major whey protein was isolated by CM-cellulose chromatography and on columns of Sephadex G-100. 4. The amino acid composition of the protein, taken in conjunction with its other properties, indicated that the major whey protein in guinea-pig milk is homologous with cow α-lactalbumin and that β-lactoglobulin is absent from guinea-pig milk. 5. Guinea-pig α-lactalbumin, which was obtained crystalline, had mol.wt. 15800, N-terminal lysine and C-terminal glutamine.     

The isolation and partial characterization of α1-proteinase inhibitor from the serum of the ostrich (struthio camelus)

• 1.1. Native and cleaved α₁-proteinase inhibitor was purified from ostrich serum using Sepharose-blue dextran chromatography, ammonium sulfate precipitation and ion exchange chromatography on DEAE-Toyopearl 650 M at pH 8.8 and 6.5.
• 2.2. Ostrich α₁PI displayed M_r values of 68,100 using gradient PAGE and 66,200 using Ferguson plots.
• 3.3. Isoelectric focusing of ostrich α₁-PI in the pH range 3–10 revealed pi values of 4.84 and 4.91, and in the pH range 4–6 the characteristic microheterogeneity observed for mammalian α₁-PIs was displayed.
• 4.4. The presence of sialic acid, hexoses and hexosamines was detected using chemical methods, but were found in much lower quantities as compared to α₁-PIs of other species.
• 5.5. Western blot analysis demonstrated a positive reaction between the native and cleaved ostrich α₁-PIs and the antibodies to the ostrich α₁-PIs raised in rabbits. No cross-reactivity was demonstrated by Western blot analysis between human α₁-PI and antibodies to ostrich α,-PI.
• 6.6. The inhibitory effect of α₁-PI on elastase and chymotrypsin was also investigated.

     

Isolation and characterization of β-glucosidase from the cytosol of rat kidney cortex

A procedure is described for the preparation of extensively purified β-d-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the β-glucosidase in the high speed supernatant (100 000 × g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. β-Glucosidase activity co-chromatographs with β-d-galactosidase, β-d-fucosidase, α-l-arabinosidase and β-d-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of β-glucosidase, respectively. The specific activity of the apparently homogeneous β-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-β-d-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6–7) and heat lability, and co-migrate on polyacrylamide disc gels at ph 8.9 (R_F, 0.67). β-Glucosidase activity is inhibited competitively by glucono-(1 → 5)-lactone (K_I, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5′-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of β-d-glucose, it will not hydrolyze xylosyl-O-serine, β-d-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000–58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of β-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convulated tubule. The enzyme is also present in relatively large ammounts in the villus cells, but not crypt cells, of the intestine. the physiological subtrates and function of the enzyme are unknown.     

α-Crystallin. The isolation and characterization of distinct macromolecular fractions

alpha-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6x10(5)-9x10(5), 0.9x10(6)-4x10(6) and greater than 10x10(6). The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65-75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5x10(3) and 22.5x10(3) were observed for all alpha-crystallin fractions.     

Structural characterization and promoter activity analysis of the γ-kafirin gene from sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions ?192 and ?476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.     

The isolation and characterization of β-glucogallin as a novel aldose reductase inhibitor from Emblica officinalis

Diabetes mellitus is recognized as a leading cause of new cases of blindness. The prevalence of diabetic eye disease is expected to continue to increase worldwide as a result of the dramatic increase in the number of people with diabetes. At present, there is no medical treatment to delay or prevent the onset and progression of cataract or retinopathy, the most common causes of vision loss in diabetics. The plant Emblica officinalis (gooseberry) has been used for thousands of years as a traditional Indian Ayurvedic preparation for the treatment of diabetes in humans. Extracts from this plant have been shown to be efficacious against the progression of cataract in a diabetic rat model. Aldose reductase (ALR2) is implicated in the development of secondary complications of diabetes including cataract and, therefore, has been a major drug target for the development of therapies to treat diabetic disease. Herein, we present the bioassay-guided isolation and structure elucidation of 1-O-galloyl-β-D-glucose (β-glucogallin), a major component from the fruit of the gooseberry that displays selective as well as relatively potent inhibition (IC(50) = 17 μM) of AKR1B1 in vitro. Molecular modeling demonstrates that this inhibitor is able to favorably bind in the active site. Further, we show that β-glucogallin effectively inhibits sorbitol accumulation by 73% at 30 μM under hyperglycemic conditions in an ex-vivo organ culture model of lenses excised from transgenic mice overexpressing human ALR2 in the lens. This study supports the continued development of natural products such as β-glucogallin as therapeutic leads in the development of novel therapies to treat diabetic complications such as cataract.     

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian alpha-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the beta-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An alpha-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.     

Isolation and characterization of A new β-melanotropin from horse pituitary glands

A new β-melanotropin was isolated from horse pituitaries and its primary structure has been determined. The amino acid sequence of the new peptide differed from that of β-melanotropin in the deletion of the NH₂-terminal aspartic acid. Thus, it is designated as [Des-Asp¹]-β-melanotropin. It possessed higher lipolytic activity when compared with β-melanotropin.     

The isolation and properties of δ-tocotrienol from Hevea latex

1. delta-Tocotrienol (8-methyltocotrienol) was isolated from the latex of Hevea brasiliensis. This new member of the tocopherol family is a pale-yellow oil at room temperature. 2. The properties of delta-tocotrienol are very similar to those of delta-tocopherol and the small differences can be explained by the change in side chain. 3. The ultraviolet and infrared spectra of delta-tocotrienol were determined and a conversion factor for use with the Emmerie-Engel reaction was worked out. Details are given for the chromatography of delta-tocotrienol on thin layers (adsorption and partition) and reversed-phase paper, and the nitroso derivatives were formed. 4. An ethyl carbonate ester of delta-tocotrienol was prepared and compared with a similar ester of delta-tocopherol. 5. Hydroxymethylation of delta-tocotrienol followed by reduction gave beta-tocotrienol as a major product.     

Mammalian epidermal keratin Isolation and characterization of the α-helical proteins from newborn rat

Neutral buffer-insoluble proteins extracted from newborn rat epidermis with alkaline urea have been purified by chromatography on Sephadex G-150 columns run in the presence of sodium dodecyl sulfate. Two proteins with apparent molecular weights of 60 000 and 68 000, respectively have been isolated and characterized. Spectropolarimetric studies show both of them to be α-helical in contrast to the non-helical heavier and lighter species also solubilized with alkaline urea. The amino acid composition of the two proteins, their electrophoretic behavior and their immunological characteristics are essentially identical. Both proteins appear to be major constituents of rat epidermal tonofilaments.     

Presence of α-neo-endorphin-like immunoreactivity in the posterior lobe of the pituitary gland

α-neo-endorphin-like immunoreactivity was demonstrated in the nerve fibers and Herring's bodies in the posterior lobe of rat pituitary glands by an indirect immunoperoxidase method using α-neo-endorphin-antiserum. The number of α-neo-endorphin positive fibers and Herring's bodies did not decrease in the sections in which α-neo-endorphin-antisera pretreated with oxytocin, ADH and leu-enkephalin were used as primary antisera. In view of the reports that met-enkephalin, leu-enkephalin and dynorphin were present in the posterior lobe of the pituitary gland, this finding suggested that there were four kinds of opiate-like peptides in the posterior lobes of the pituitary gland. Furthermore, by staining alternately 3he serial sections of the rat pituitary glands with ADH and α-neo-endorphin-antisera, it was revealed that α-neo-endorphin-positive Herring's bodies were identical to a large number of ADH positive Herring's bodies. This finding, together with the observation that morphine injection caused ADH release, suggested that α-neo-endorphin may play an important role in the regulation of ADH release.     

Differential control of β-endorphin/β-lipotropin secretion from anterior and intermediate lobes of the rat pituitary gland in vitro

The secretion of immunoreactive β-endorphin (β-END_i) and β-lipotropin (β-LPH_i) by neurointermediate lobes (NIL) and anterior lobe (AL) cells of the rat pituitary gland was studied in an $\text{in vitro}$ superfusion system. Peptides were characterized by gel chromatography on Sephadex G-50 and by two radioimmunoassays: a β-LPH assay in which β-END did not crossreact (β-LPH_i) and a β-END/β-LPH assay in which β-END and β-LPH showed full crossreactivity (β-END_i/β-LPH_i).$\text{Intermediate lobe}$. The spontaneous secretion of β-END_i/β-LPH_i by the NIL was 1–2 ng/min/lobe. Chromatography showed that 97% of this β-END_i/β-LPH_i eluted at the position of β-END. Dopamine inhibited the spontaneous secretion of β-END and the dopamine-receptor blocker domperidone prevented this inhibition. Isoprenaline caused a 3–4 fold stimulation of the secretion of β-END. The β-adrenergic receptor blocker propranolol abolished this stimulation. Hypothalamic extract, lys-vasopressin, 5-hydroxytryptamine and histamine were ineffective in changing the spontaneous secretion of β-END_i/β-LPH_i.$\text{Anterior lobe}$. The spontaneous secretion of β-END_i/β-LPH_i by AL cells was 0.15–0.20 ng/min/10⁵ cells. Chromatography revealed that about 70% of this material behaved like β-LPH, 30% behaved like β-END. Hypothalamic extract and lys-vasopressin induced a 3–5 fold increase in the secretion of both β-END and β-LPH. Catecholamine, 5-hydroxytryptamine and histamine were ineffective in changing the spontaneous secretion of β-END_i/β-LPH_i.These results indicate that β-END is the predominant β-LPH-related peptide secreted by the intermediate lobe and that its secretion is inhibited via a dopaminergic receptor mechanism and stimulated via a β-adrenergic receptor mechanism. The secretion of β-END and β-LPH by the anterior lobe is not affected by catecholamines but is stimulated by CRF and vasopressin.