Chromatographic separation of thyroid stimulating hormone from luteinizing hormone

A new preparative scale procedure is described for the efficient purification of Thyroid stimulating hormone (TSH) from buffalo pituitary glands. The methodology involves the use of hydrophobic interaction chromatography for the separation of TSH from LH which is more abundant than TSH in the pituitary gland. The two hormones were isolated from the pituitary extract by ammonium sulphate precipitation and were then separated on a Phenyl-Sepharose column. The fractions obtained from Phenyl-Sepharose chromatography were assessed using a Uno-Q column on FPLC where enrichment of the TSH in the PS-2 fraction was evident. The final removal of the trace amounts of the LH was done by ion exchange chromatography in tandem on CM-Sephadex and DEAE-Sephacel columns. All the chromatography fractions were assessed in a heterologous direct binding ELISA using bTSH and buLH as the reference hormones and anti ovine TSH beta and anti bovine LH beta as the antisera. The purified fraction (DEAE-0.5 M peak) when checked exhibited no LH immuno reactivity in a western blot against anti bovine LH beta antiserum. Forty two milligrams of a 200 fold purified buTSH per kg wet glands was obtained

Proteostatic Tactics in the Strategy of Sterol Regulation

In eukaryotes, the synthesis and uptake of sterols undergo stringent multivalent regulation. Both individual enzymes and transcriptional networks are controlled to meet changing needs of the many sterol pathway products. Regulation is tailored by evolution to match regulatory constraints, which can be very different in distinct species. Nevertheless, a broadly conserved feature of many aspects of sterol regulation is employment of proteostasis mechanisms to bring about control of individual proteins. Proteostasis is the set of processes that maintain homeostasis of a dynamic proteome. Proteostasis includes protein quality control pathways for the detection, and then the correction or destruction, of the many misfolded proteins that arise as an unavoidable feature of protein-based life. Protein quality control displays not only the remarkable breadth needed to manage the wide variety of client molecules, but also extreme specificity toward the misfolded variants of a given protein. These features are amenable to evolutionary usurpation as a means to regulate proteins, and this approach has been used in sterol regulation. We describe both well-trod and less familiar versions of the interface between proteostasis and sterol regulation and suggest some underlying ideas with broad biological and clinical applicability

A simple method for isolation of three reproductive hormones together from the same batch of freshly frozen buffalo pituitaries collected from abattoirs

Pseudo-affinity chromatography using Concanavaline-A-Sepharose, the immobilized form of the well known plant lectin, was used to isolate the glycoprotein fraction of buffalo pituitary extract. The lectin bound proteins were eluted and stored lyophilized. This isolate was analyzed by ELISA and by hormone specific bioassays in rats to obtain potency estimates of Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Thyroid Stimulating Hormone (TSH) content. Hopefully this isolate will be useful in inducing super ovulation in animals including buffaloes

Pseudo-affinity chromatographic approach to probe heterogeneity in buffalo pituitary luteinizing hormone: probable pseudolectin-like behavior of immobilized Cibacron Blue 3GA

The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and β subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar–dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue

Interaction between cibacron blue F3GA and Luteinizing hormone: a chromatographic investigation

Luteinizing hormone which was bound to Cibacron Blue F3GA could not be eluted with 10mM NAD. Bound LH could be eluted partially (up to 5%) with 80% ethylene glycol and rest with 50mM phosphate buffer pH 7.5 containing 1M NaCl. Each of the bound fractions could also be sub-fractionated with differential elution with a gradient of ethylene glycol or NaCl. This indicated that pituitary LH was a mixture of two different sub-populations of LH, one which interacts with CB predominantly via hydrophobic interactions and the other via electrostatic interaction. In the case of subunits of LH that occur in free state in pituitaries, approximately half of the bound subunits interacted with CB column predominantly via hydrophobic interactions whereas the other half interacted via electrostatic force. It is concluded that differences in glycan content, composition and structure could be the cause for differential binding of buLH and its free subunits to the CBA column

Direct and essential function for Hrd3 in ER-associated degradation

The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells

Mechanistic investigations of the dual activity of Gonadotropins using target tissue cAMP level as a response parameter

Gonadotropins like human chorionic gonadotropin (hCG) and pregnant mare serum gonadotropin (PMSG) exhibit dual activities on target tissues. The status of cAMP as a second messenger in these activities has been examined experimentally. Direct correlation with target tissue cAMP concentration, has been observed in the case of thyrotropic effect of hCG on thyroid tissue and the ovarian ascorbic acid depletion effect of hCG on superovulated immature rat ovaries. Marked difference in the response of buffalo tissues in comparison to rat tissues has been observed. No correlation could be observed between cAMP levels and ovarian ascorbic acid content in the case of PMSG action. Surprisingly a peptide derived from buffalo Prolactin appears to have antigonadotropic effect as far as ovarian ascorbic acid content is concerned

Direct and essential function for Hrd3 in ER-associated degradation

The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells

Direct involvement of Hsp70 ATP hydrolysis in Ubr1-dependent quality control.

Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone-dependent ubiquitination by the action of the E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70's role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of Sse1, and the action of J-proteins are all needed for Ubr1-mediated quality control. To discern whether chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage in which efficiently folded proteins are spared, while slow-folding or nonfolding proteins are iteratively tagged with ubiquitin for subsequent degradation