The effect of hypophysectomy and hypophysis-transplantation on the secretion of gut glucagon immunoreactivity and gut glucagon-like immunoreactivity in depancreatized dogs

The role of hypophysis in the regulation mechanism of the secretion of gut glucagon immunoreactivity (gut GI) that was measured using C-terminal specific glucagon antiserum after pancreatectomy, and gut glucagon-like immunoreactivity (gut GLI) that was obtained by subtracting GI from total glucagon-like immunoreactivity (total GLI) which was measured using non-specific glucagon antiserum, was investigated in depancreatized dogs. Plasma glucose, gut GI and gut GLI levels were found to increase in totally depancreatized dogs. The former two showed a significant decrease after hypophysectomy, and were reversed by the hypophysis-transplantation, while gut GLI was not affected either by hypophysectomy or hypophysis-transplantation. Intramuscular injections of human growth hormone (HGH) or adrenocorticotropic hormone-Z (ACTH-Z) to depancreatized-hypophysectomized dogs had no effect on plasma glucose level or gut GI. It is concluded that hypophysis may promote the secretion of gut GI after pancreatectomy, but not of gut GLI. Gut GI seems to regulate plasma glucose level after pancreatectomy. However, the precise regulation mechanism of gut GI by the hypophysial hormone after pancreatectomy is not clarified yet.     

Characterization of a heat stable, 12,000 Da, glucagon-like immunoreactive (GLI) peptide from porcine ileum that stimulates hepatic glucose production in vivo and in vitro

Glucagon-like immunoreactivity (GLI) was extracted from porcine ileal mucosa with boiling 2 M HAc followed by elution on DEAE A-50 and fractionation on Sephadex G-50 F. Intact GLI was isolated from mesenteric blood by fractionation of several plasma samples from a mesenteric vein of a dog on Sephadex G-50 M followed by refractionation of the pooled GLI from these columns on G-50 F. Analysis of the semipurified mucosal and plasma GLI on Sephadex G-50 SF, eluted with 0.1 M Tris/HCl + 8 M urea, pH 7.5, demonstrated a single, sharp peak of GLI with a relative molecular mass (Mr) between 12,000 and 13,000. Electrophoresis on PAGE gels at acid pH with 2 M urea demonstrated a single charged GLI species in both the plasma and mucosal fractions. Stimulation of the release of GLI from a loop of ileum isolated in situ in an anesthetized dog followed removal of the known sources of glucagon (stomach, pancreas, and duodenum) resulted in an immediate and sustained increase in hepatic glucose production. The isolated GLI from ileal mucosa (5 X 10(-8) M) stimulated gluconeogenesis from 10 mM [14C]alanine in hepatocytes isolated from fed rats. The stimulation was equal to 25% of the maximal stimulation observed with 10(-8) M glucagon. These experiments demonstrate that the ileum synthesizes and secretes a GLI peptide with a Mr of approximately 12,000 that stimulates hepatic glucose production in vivo and in vitro.     

Effect of phenformin on the response of plasma intestinal glucagon-like immunoreactivity (GLI) to oral glucose in gastrectomized subjects.

The effect of phenformin (DBI) on the plasma intestinal glucagon-like immunoreactivity (GLI) and pancreatic glucagon (IRG) responses to oral and intravenous glucose loads were studied in 26 gastrectomized subjects, using a cross-reacting and an IRG-specific anti-serum. The drug produced no significant changes in fasting GLI and IRG levels. Thirty minutes after oral glucose alone, the total GLI level rose to a peak of 1.55 +/- 0.17 ng/ml in the untreated subjects and to a maximum level of 1.67 +/- 0.18 ng/ml in the DBI-pretreated subjects. However, the mean GLI levels obtained 120 and 180 min after oral glucose were significantly higher after treatment with DBI. The blood sugar and IRI responses to oral glucose were lowered significantly by DBI pretreatment. DBI did not alter the glucose, IRI, IRG and GLI response to intravenous glucose. These results suggest that the release of intestinal GLI is not related to the intestinal absorption of glucose.     

A flow-calorimetric study of the binding of iodine to amylodextrin fractions

Acid hydrolyzates of waxy-maize starch were separated to give Fractions I, II, and III [T. Watanabe, and D. French, Carbohydr. Res., 84 (1980) 115-123]. Watanabe and French suggested that Fraction II, which contains approximately 25 D-glucose residues including an alpha-D-(1----6)-linked branch, has a double helical structure. In the present study, the thermodynamics of binding of iodine to Fractions II and III, and debranched Fraction II (Fraction II') was measured by isothermal-flow calorimetry. If four binding sites for Fraction II and two for Fractions II' and III are assumed, the standard free-energy changes, delta Gb0, for the binding of I2 are -18.5, -18.8, and -18.4 kJ X (mol I2)-1, and the enthalpy changes, delta Hb, are -28.4, -24.7, and -26.9 kJ X (mol I2)-1, respectively. The similarity of these values for the three fractions indicates that the conformation of Fraction II is essentially the same as those of Fractions II' and III, and that Fraction II, therefore, does not have a double helical structure in solution. The values for delta Gb0 are approximately 15 kJ X mol-1 less negative, and those for delta Hb approximately 40 kJ X mol-1 less negative than published values for the starch-I2 complex. These differences are due to the relatively very short D-glucose chains in the amylodextrin fractions employed in the present work.     

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.     

Synthesis of low-phenylalanine polypeptides

Low-phenylalanine-peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by alpha-chymotrypsin then followed by carboxypeptidase-A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight greater than 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soy bean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.     

Conversion of Anhydride to Thiolactone in Biotin Synthesis

This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 °C for 6 hours after conditioning at 4 °C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500–1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.     

Sourness-suppressing peptides in cooked pork loins

This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 degrees C for 6 hours after conditioning at 4 degrees C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500-1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.     

A case with glucagonoma syndrome--heterogeneity of glucagon and insulin

The heterogeneity of glucagon and insulin in plasma and tissue extracts from a 57-year-old female with glucagonoma syndrome with surgically and autopsy verified islet-cell tumors was studied by Bio-Gel P-10 filtration. The preoperative plasma immunoreactive glucagon (IRG) level was 20.2 ng/ml, and plasma glucagon-like immunoreactivity(GLI) 25.8 ng/ml. The column chromatography of the preoperative plasma revealed three or four IRG components and four GLI components. Among these, peak II, the large glucagon immunoreactivity (LGI) peak, considered a candidate for proglucagon, was prominent, along with peak III. The resected metastatic liver tumor contained an enormous amount of IRG and an appreciable amount of immunoreactive insulin (IRI), indicating that the elevated plasma IRG was mainly of tumor origin. The IRG pattern of the tumor tissue extract revealed a small quantity of IRG in peaks I and II, and a large amount in peak III; control pancreatic tissue extract manifested a similar elution pattern. The IRI elution pattern of the tumor tissue extract revealed two major IRI peaks which migrated close to the elution volume of cytochrome C and insulin, respectively. This is a quite different pattern from the control pancreatic tissue extract in which the RI peak was localized in the elution volume of the insulin. We conclude that the present metastatic liver tumor produced not only enormous amounts of glucagon but heterogeneous peptides which contained immunological insulin determinants within their.     

A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.     

Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides

Synopsis The coloured components in the high-iron diamine dye bath were separated into three fractions using column chromatography on Sephadex G-10. These fractions were called Fraction I, II and III in order of their emergence from the column. From atomic absorption measurements, part of Fraction I was found to be free of iron. Most of Fraction II and the whole of Fraction III contained only trace amounts of iron. Therefore, the three Fractions were investigated further. All experiments were carried out at pH 1.4 (corresponding to the pH of the original high-iron diamine bath).Fraction I was violet, Fraction II red-violet and Fraction III aniline-red; the extinction maxima in the visible region were 560, 526 and 540 nm respectively. On electrophoresis, the Fractions were not quite homogeneous, although most of Fractions I and II migrated in the same front and much faster than Fraction III. The high-iron diamine solution separated into two main fractions, one of which corresponded in colour and velocity to Fractions I and II and the other to Fraction III.In histochemical experiments, Fractions I, II and III bound to tissue sites containing sulphated mucosaccharides or nucleic acids; from histochemical enzyme digestion tests or by using purified materials in spot tests on cellulose acetate membrane, it was confirmed that the diamines were bound to RNA and DNA. However, when ferric chloride was added to any of the Fractions in an amount corresponding to that in the original high-iron diamine dye bath, the binding to tissue sites containing nucleic acids was inhibited but the reaction with sulphated mucosubstances was not affected. Also, in the presence of added ferric chloride, the anomalous binding of Fraction I to carboxyl groups of mouse sublingual gland sialomucin was prevented.It is concluded that ferric chloride in the high-iron diamine dye bath prevents diamine complexes from binding with nucleic acids, and apparently with carboxymucins too. Further, this conclusion substantiates our previous observations of the central role ferric chloride plays in making the histochemical high-iron diamine technique specific for sulphated mucosubstances.     

Isolation and partial characterization of the major apolipoprotein component of bovine serum high density lipoprotein.

The delipidated protein component of bovine serum high density lipoprotein was fractionated by gel filtration on a Sephadex G-150 column (equilibrated with buffer containing 6 M urea) into three fractions: I, II and III. Fractions I and II together constitute 88% of all the protein weight of bovine high density lipoprotein, whereas fraction III accounts for the remaining 12%. Analysis of the fractions by sodium dodecyl sulfate-polyacrylamide electrophoresis reveals that fraction I consists mostly of aggregated forms of fraction II and some higher molecular weight species, probably irreversible aggregates of fraction II. The irreversible aggregates are apparently formed during the delipidation procedure or upon aging of the lipoprotein. The major protein component of the high density bovine lipoprotein is found in fraction II; it has a molecular weight of 27 000 plus or minus 1500 and appears to be homogeneous by several physicochemical criteria. The amino acid composition of fractions I and II are essentially identical; their spectral properties, including absorption, fluorescence, and circular dichroism spectra, are similar; however, fraction I appears to contain traces of oxidized lipid and more secondary alpha-helical organization than fraction II. By comparison with the intact lipoprotein, which contains about 65% of alpha-helical structure, fractions I and II have diminished alpha-helical organization, 55% and 43%, respectively. Fraction III, on sodium dodecyl sulfate-polyacrylamide electrophoresis, separates into two protein bands of equivalent intensity, having molecular weights around 13 000 and 11 000. Fraction III is markedly distinct from the other two, in amino acid composition and spectral properties, especially in its red-shifted fluorescence and very low content of alpha-helical structure. The protein composition of bovine serum high density lipoprotein is compared with recently published results for high density lipoprotein apoproteins of man, chimpanzee, rhesus monkey, pig and rat. Similarities and differences are discussed in terms of possible evolutionary and functional factors.     

The use of perfused rat intestine to characterise the glucagon-like immunoreactivity released into serosal secretions following stimulation by glucose.

Isolated perfused intestine of rat was used to demonstrate the glucose-stimulated release of glucagon-like immunoreactivity (GLI) into serosal secretions. The released GLI was characterised using immunoaffinity chromatography on columns of immobilised antibodies specific for the N (residues 1 to 18) and for the C (residues 19-29) terminal portions of glucagon followed by gel-filtration. The immunoreactivity was present in a variety of molecular species. These include a large GLI which has a molecular weight about 12000 and binds to antibodies specific for the N-terminal portion of glucagon and two polypeptide fractions with molecular weight closer to that of glucagon. While one fraction of the small GLI boun both to antibodies specific for the C-terminal and N-terminal portions of glucagon the other bound only to the former antibodies. The relevance of these findings to the origins of circulating GLI and the possible precursor relationship between large and other forms of GLI is discussed.     

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.     

Pancreatic hormones and plasma glucose: regulation mechanisms in the goose under physiological conditions. I. Pancreatectomy and replacement therapy

Measurements of plasma GLI and IRI in normal fasting geese, before and during constant I.V. infusion of saline, gave GLI/I ratios of 1.32 +/- .07 and 1.34 +/- .03 (w/w). As total pancreatectomy markedly reduces the pancreatic hormone level, leading to a mortal hypoglycaemia, we attempted to maintain plasma glucose within the normal range by constant I.V. infusion of glucagon and insulin into operated animals. The results as follows: 1. Blood glucose levels can be maintained within the normal range during experiments lasting 6 or more hours with a constant G/I ratio. 2. The G/I ratio obtained in operated animals (.96 +/- .12) is near to, but significantly lower (p less than .005) than, the GLI/I ratio measured in normal animals. This difference may be explained by the presence of a small amount of circulating gut GLI in the 2nd group.     

Localization of androgen receptors in ram epididymal principal cells

Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 10(6) total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.     

Differential Expressions of Two Types of Alkaline Phosphatase during Developmental Growth of Embryoid Bodies of Mouse Teratocarcinoma OTT-6050 In Vitro

Ascites teratocarcinoma OTT-6050 is a totipotent tumor line producing indefinitely the simple type of embry-oid bodies (EBs). In culture with fetal calf serum (FCS) in Eagle's minimal essential medium (MEM), these EBs show developmental growth, only in which some differentiative events result. EBs also show this developmental growth in MEM supplemented with two fractions of FCS separated with a Amicon PM 10 membrane, i.e. a low molecular weight Fraction L (mol. wt. less than 10,000) and a high molecular weight Fraction H (mol. wt. more than 10,000). Fraction H is necessary for the survival of EBs in vitro. Fraction L enhances the uptake of ³H-thymidine into EB cells with increase in the Vmax , but no change in the K m. On culture of EBs with both Fractions, a marked bimodal increase in alkaline phosphatase (ALPase) activity is seen on day 1–2 and 4, resulting from the differential expressions of two electrophoretically distinct ALPases (Bands I and II). The differential expressions of ALPase are also observed cytochemically, one activity being on the inner cells and the other on the surrounding cells of EBs. From the cytochemical similarity of ALPase activity to that of normal mouse embryos, Band I ALPase is inferred to be the epiblast (developmentally totipotent stem cell)-type and Band II ALPase to be the distal (parietal) endoderm-type.     

Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?     

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

A glycosaminoglycan activator of lipoprotein lipase in human plasma

By use of ion exchange chromatography we have isolated two discrete classes of “free” glycosaminoglycans (GAG) from human plasma. The GAG fractions were tested for their effects on two lipoprotein lipase (LPL) enzyme systems containing an apolipo-protein C-II activated emulsion as the triglyceride substrate and bovine serum albumin as the free fatty acid acceptor. The low-charge GAG (Fraction I) had essentially no effect on the LPL reaction. The high-charge GAG (Fraction II) stimulated the LPL reaction 100 to 300%. The GAG composition of each fraction was investigated with chemical and enzymatic techniques. Fraction I consisted of low-charge chondroitin sulfate noncovalently bound to protein. Fraction II consisted of a mixture of high-charge GAG non-covalently bound to protein. Degradation with nitrous acid eliminated the ability of high-charge GAG to stimulate LPL. This and other evidence suggests that the high-charge GAG in human plasma responsible for LPL activation is heparan sulfate (HS). We suggest that plasma HS may modulate triglyceride clearance mechanisms $\text{in vivo}$ by its interaction with LPL.